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PATHOLOGY EXAMINATION ANSWERS

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Q1 (20 Marks)

Aetiology of Acquired Valvular Lesions of the Heart

Aetiological Classification

1. Rheumatic Heart Disease (Most Common Worldwide)

• Follows Rheumatic Fever (Group A beta-hemolytic Streptococcus pharyngitis)
• Mitral valve most commonly affected, followed by aortic > tricuspid > pulmonary
• Pathology: fibrous thickening, commissural fusion, chordal shortening, calcification
• Results in: mitral stenosis (most characteristic), combined mitral stenosis + regurgitation

2. Degenerative / Calcific Disease

• Senile calcific aortic stenosis: Most common cause of AS in elderly (>70 yrs); calcium deposits in cusps, no commissural fusion
• Mitral annular calcification: Mainly causes regurgitation; common in elderly women
• Myxomatous degeneration (Mitral Valve Prolapse): Excess proteoglycans in spongiosa, billowing leaflets causing mitral regurgitation; Marfan syndrome is a classic predisposing condition

3. Infective Endocarditis

• Bacterial/fungal destruction of cusps, perforations, vegetations
• Causes acute severe regurgitation (aortic or mitral most commonly)
• May heal with fibrosis and stenosis

4. Ischaemic Heart Disease

• Papillary muscle dysfunction or rupture following MI causes acute mitral regurgitation
• Ischaemic annular dilatation causes functional MR without structural leaflet damage

5. Carcinoid Heart Disease

• Right-sided valves affected (pulmonary and tricuspid)
• Serotonin (5-HT) causes fibrous plaques - "carcinoid plaques" - on endocardial surface
• Results in tricuspid regurgitation and pulmonary stenosis

6. Drug-Induced Valvulopathy

• Ergot alkaloids (methysergide, ergotamine), fenfluramine-phentermine, pergolide, cabergoline
• Fibrous endocardial plaques similar to carcinoid

7. Connective Tissue / Autoimmune Diseases

• SLE: Libman-Sacks endocarditis - non-bacterial thrombotic vegetations on both surfaces of mitral/aortic valves (antiphospholipid syndrome)
• Rheumatoid arthritis: granulomatous nodules on valves
• Ankylosing spondylitis: aortic regurgitation

8. Iatrogenic / Procedural

• Radiation-induced valvular fibrosis (especially after mantle field radiotherapy)
• Prosthetic valve dysfunction (structural, thrombosis, paravalvular leak)

9. Rare Causes

• Syphilitic aortitis: aortic regurgitation due to root dilatation
• Hypereosinophilic syndrome / Loeffler endocarditis
• Whipple disease: right-sided valves

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Infective Endocarditis (IE)

Definition

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Etiopathogenesis

Step 1: Endothelial Damage / Non-Bacterial Thrombotic Endocarditis (NBTE)

• Normal endocardium is resistant to bacterial adhesion
• Turbulent blood flow (due to valvular disease, congenital defects, prosthetic valves, IDU-related right-sided jets) causes mechanical endothelial damage
• Damaged endothelium exposes subendothelial collagen and tissue factor
• This triggers platelet aggregation and fibrin deposition, forming a sterile thrombus (NBTE / marantic endocarditis) - the critical precursor lesion

Step 2: Bacteraemia

• Transient bacteraemia is common: dental procedures, GI/GU instrumentation, IV drug use, intravascular catheters, skin infections
• High-density bacteraemia with virulent or adherent organisms is required for IE to develop

Step 3: Bacterial Adherence and Colonisation

• Bacteria must adhere to the NBTE lesion
• Virulence factors promoting adhesion:
  • S. aureus: fibronectin-binding proteins (FnBP), clumping factor A (ClfA), MSCRAMM family adhesins
  • Viridans streptococci: dextran production, FimA, glucan-binding proteins
  • Enterococcus, S. bovis, HACEK group: various adhesins
• Fibronectin acts as a bridge between bacteria and platelet-fibrin thrombus

Step 4: Vegetation Formation and Propagation

• Bacteria proliferate within the vegetation, protected from host immunity
• Further platelet deposition and fibrin layering buries bacteria (metabolically inactive inner core - explains why prolonged antibiotics needed)
• Vegetation size increases
• Tissue destruction: collagenases, hyaluronidases, proteases destroy valve leaflet
• Periannular abscess formation: direct extension of infection to adjacent structures

Step 5: Systemic Complications

• Embolic: Fragments break off and cause:
  • Systemic emboli (aortic/mitral IE): stroke, splenic infarcts, renal infarcts, septic emboli
  • Pulmonary emboli (tricuspid IE in IDU)
  • Roth spots (retinal), Janeway lesions (non-tender embolic infarcts), Osler nodes (immune complex deposition)
• Immune complex deposition: Glomerulonephritis, arthritis, vasculitis
• Metastatic infection: Mycotic aneurysms (weakened vessel walls from infected emboli or immune complex deposition)

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Risk Factors for IE

Cardiac Risk Factors (Predisposing Structural Lesions)

Non-Cardiac Risk Factors

• IV drug use: Major risk factor for right-sided (tricuspid) IE; S. aureus predominant
• Intravascular devices: Central venous catheters, pacemaker/ICD leads (device-related IE)
• Haemodialysis: Arteriovenous fistula, tunneled catheters
• Poor dental hygiene / dental procedures: Source of viridans streptococcal bacteraemia
• Immunosuppression: HIV, chemotherapy, immunosuppressants
• Skin infections / breaks: S. aureus entry
• Gastrointestinal procedures / malignancy: S. bovis (S. gallolyticus) bacteraemia - associated with colonic neoplasia

Microbial Risk Factors

• S. aureus: Most virulent; can infect normal valves; #1 cause in developed countries
• Viridans group streptococci: Subacute IE on damaged valves
• Enterococcus faecalis: Associated with GI/GU procedures
• Coagulase-negative Staphylococci: Prosthetic valve IE
• HACEK group (Haemophilus, Aggregatibacter, Cardiobacterium, Eikenella, Kingella): Slow-growing, culture-negative
• Fungi (Candida, Aspergillus): IDU, immunocompromised, prosthetic valves

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Pathology

Macroscopic Features

• Vegetations: The hallmark lesion; irregular, friable, tan-grey masses adherent to valve leaflets
  • Location: typically on the atrial surface of AV valves and ventricular surface of semilunar valves (low-pressure side)
  • Size: variable, 0.5 cm to several centimetres
  • Right-sided vegetations (tricuspid) in IDU tend to be large and bulky
• Valve destruction: Perforations, erosions, aneurysm of cusp/leaflet
• Periannular abscess (ring abscess): Direct extension, especially in aortic valve IE; may fistulise
• Chordal rupture: Especially in mitral valve IE
• Myocardial abscess: Extension to myocardium
• Pericarditis: In S. aureus IE or periannular abscess perforation

Microscopic Features

• Vegetation: fibrin, platelets, bacteria, numerous neutrophils
• Underlying valve leaflet: coagulative necrosis, destruction of collagen
• Heavy bacterial colonies visible within vegetation
• Abscess formation with neutrophils

• Vegetation: fibrin, platelets, granulation tissue, sparse organisms
• Underlying valve: chronic inflammatory cells, fibroblast proliferation
• Healed vegetations: organized fibrous tissue, calcification, new vessel formation

Complications (Pathological Basis)

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Modified Duke Criteria (Diagnostic Framework)

Major Criteria

1. Positive blood cultures (at least 2 of typical IE organisms, or persistently positive, or single positive for Coxiella burnetii)
2. Positive echocardiographic findings: vegetation, abscess, prosthetic valve dehiscence, or new valvular regurgitation

Minor Criteria

1. Predisposing heart condition or IV drug use
2. Fever >38°C
3. Vascular phenomena (emboli, mycotic aneurysms, Janeway lesions, conjunctival haemorrhages)
4. Immunological phenomena (glomerulonephritis, Osler nodes, Roth spots, rheumatoid factor)
5. Microbiological evidence not meeting major criteria

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Diagnostic Investigations

Blood Cultures (Most Important Investigation)

• Method: 3 sets from separate venipuncture sites, 30 min apart, before antibiotics
• Volume: 10 mL per bottle (adult) - increases yield
• Timing: Before any antibiotic administration
• Yield: 90-95% positive in native valve IE if done correctly
• Organisms: S. aureus and streptococci identified within 24-72 hours
• HACEK organisms: Culture-negative - require prolonged incubation (up to 2 weeks) or molecular methods
• Culture-negative IE: Due to prior antibiotics, fastidious organisms (Bartonella, Coxiella, Tropheryma whipplei, Brucella)

Echocardiography

• Transthoracic Echo (TTE): First-line; sensitivity 40-63% for vegetations (S. aureus IE, prosthetic valves - lower sensitivity)
• Transesophageal Echo (TEE): Sensitivity 87-100%; mandatory when:
  • TTE non-diagnostic but IE suspected
  • Prosthetic valve IE
  • S. aureus bacteraemia
  • Suspected periannular complications
  • Assessment before/after surgery
• Findings on echo: Oscillating masses (vegetations), abscesses, fistulae, new regurgitation, dehiscence

Laboratory Investigations

• CBC: Leucocytosis (acute IE), anaemia of chronic disease (subacute IE), thrombocytopenia (sepsis)
• ESR and CRP: Markedly elevated; useful for monitoring treatment response
• Rheumatoid factor: Elevated in ~50% of subacute IE (non-specific)
• Serum procalcitonin: Elevated; aids in differentiating bacteraemia from IE
• Urinalysis: Microscopic haematuria, proteinuria, red cell casts (immune complex GN)
• Renal and hepatic function: Baseline; for aminoglycoside monitoring

Imaging Investigations

• Chest X-ray: Cardiomegaly, pulmonary oedema (heart failure), multiple peripheral infarcts (right-sided IE)
• ECG: PR prolongation/heart block (periannular abscess); new LBBB
• CT scan:
  • Cardiac CT: Periannular abscess, pseudoaneurysms, fistulae
  • CT chest/abdomen/pelvis: Embolic complications (pulmonary emboli, splenic/renal infarcts)
  • CT brain: Ischaemic strokes, mycotic aneurysms
• MRI brain: More sensitive than CT for embolic cerebral lesions
• 18F-FDG PET/CT: Especially useful for prosthetic valve IE (increased metabolic activity around prosthesis)
• Radiolabelled leucocyte scintigraphy: For device-related IE diagnosis

Microbiological Investigations for Culture-Negative IE

• Serology: Coxiella burnetii (Q fever), Bartonella, Brucella, Chlamydia
• PCR: From blood or excised valve tissue - HACEK, Bartonella, Tropheryma whipplei
• 16S ribosomal RNA gene sequencing: Excised valve material
• Matrix-Assisted Laser Desorption/Ionisation (MALDI-TOF): Organism identification

Histopathological Examination (Surgical Specimens)

• Excised valve: Gram stain, culture, histology
• Histology: Vegetation with bacteria, inflammatory cells, valve destruction confirms diagnosis definitively
• Methenamine silver and PAS for fungi; Warthin-Starry for Bartonella; Ziehl-Neelsen for mycobacteria

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Q2 (20 Marks)

Classification of Bone Tumours

WHO Classification of Bone Tumours (2020)

I. Cartilage (Chondrogenic) Tumours

• Osteochondroma (solitary / hereditary multiple exostoses)
• Enchondroma (solitary / Ollier disease / Maffucci syndrome)
• Periosteal chondroma
• Chondroblastoma
• Chondromyxoid fibroma

• Atypical cartilaginous tumour / Chondrosarcoma Grade 1 (central, periosteal)

• Chondrosarcoma Grade 2 and 3
• Dedifferentiated chondrosarcoma
• Mesenchymal chondrosarcoma
• Clear cell chondrosarcoma

II. Osteogenic (Bone-Forming) Tumours

• Osteoma
• Osteoid osteoma
• Osteoblastoma

• Osteosarcoma (conventional, parosteal, periosteal, telangiectatic, small cell, secondary)

III. Fibrogenic Tumours

• Desmoplastic fibroma (intermediate)
• Fibrosarcoma (malignant)

IV. Vascular Tumours

• Haemangioma (benign)
• Epithelioid haemangioendothelioma (intermediate)
• Angiosarcoma (malignant)

V. Osteoclastic Giant Cell-Rich Tumours

• Non-ossifying fibroma
• Giant cell tumour of bone (intermediate)
• Malignant giant cell tumour

VI. Notochordal Tumours

• Benign notochordal cell tumour
• Chordoma

VII. Marrow (Round Cell) Tumours

• Ewing sarcoma / PNET
• Plasmacytoma / Multiple myeloma
• Primary bone lymphoma

VIII. Smooth Muscle Tumours

• Leiomyoma (benign)
• Leiomyosarcoma (malignant)

IX. Nerve Sheath Tumours

• Neurofibroma, schwannoma

X. Miscellaneous

• Adamantinoma
• Simple (unicameral) bone cyst
• Aneurysmal bone cyst
• Fibrous dysplasia
• Osteofibrous dysplasia
• Langerhans cell histiocytosis

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Chondrogenic Tumours - Detailed Discussion

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1. Osteochondroma (Osteocartilaginous Exostosis)

Incidence and Epidemiology

• Most common benign bone tumour (35-40% of all benign bone tumours)
• Age: 10-30 years; Male > Female (2:1)
• Hereditary Multiple Exostoses (HME): Autosomal dominant, mutations in EXT1 (8q24) and EXT2 (11p13) - tumour suppressors

Sites

• Metaphysis of long bones (distal femur, proximal tibia, proximal humerus)
• Any bone formed by endochondral ossification

Pathogenesis

• Arises from displacement of small cartilage fragments from the growth plate epiphysis
• EXT1/EXT2 mutations disrupt heparan sulphate proteoglycan synthesis, affecting Hedgehog signalling and chondrocyte differentiation

Macroscopy

• Pedunculated (stalked) or sessile (broad base) bony outgrowth
• Covered by a fibrous perichondrium and a cartilaginous cap (blue-white, glistening)
• Cap thickness <1 cm in adults (>2 cm suggests malignant transformation)
• Bony cortex is continuous with underlying bone (key diagnostic feature)

Microscopy

• Cartilaginous cap: hyaline cartilage with benign chondrocytes in lacunae, organised in rows (like normal growth plate)
• Endochondral ossification at base of cap (zone of ossification)
• Perichondrium: fibrous connective tissue
• Core: normal cancellous bone with fatty/haematopoietic marrow

Malignant Transformation

• Solitary: <1%; Multiple (HME): 5-25%
• Risk factors: increasing cap thickness, growth after skeletal maturity, pain
• Transforms to: secondary chondrosarcoma (most often, Grade 1)

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2. Enchondroma (Central Chondroma)

Epidemiology

• Second most common benign cartilage tumour
• Peak age: 10-40 years; No sex predilection
• Ollier disease: Multiple enchondromatosis (non-hereditary); 25-30% risk of malignant transformation
• Maffucci syndrome: Multiple enchondromas + soft tissue haemangiomas; >100 times malignant risk; IDH1/IDH2 mutations

Sites

• Short tubular bones of hands and feet (50-65%) - classic location
• Long bones (femur, tibia, humerus) in Ollier/Maffucci

Pathogenesis

• Residual islands of growth plate cartilage that fail to undergo endochondral ossification, arrested in medullary cavity
• IDH1 (R132) and IDH2 (R172) mutations in most enchondromas - produce 2-hydroxyglutarate (oncometabolite)

Macroscopy

• Well-circumscribed lobules of blue-white hyaline cartilage in medullary cavity
• Calcification: "popcorn" or "rings and arcs" pattern
• No cortical destruction

Microscopy

• Lobules of hyaline cartilage matrix
• Sparsely cellular; chondrocytes small and uniform in lacunae
• Minimal nuclear atypia, rare mitoses
• Calcification and endochondral ossification at periphery of lobules

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3. Chondroblastoma

Epidemiology

• Rare; peak age 10-20 years (skeletally immature); M:F = 2:1
• Essentially the only primary bone tumour confined to epiphysis in children

Sites

• Epiphysis of long bones: distal femur, proximal tibia, proximal humerus (around knee and shoulder)
• May extend into metaphysis or subarticular region

Pathogenesis

• H3.3 histone mutations: H3F3B K36M (>95% of cases) - characteristic; affects epigenetic regulation
• Considered neoplastic

Macroscopy

• Lobulated, pink-red to grey, haemorrhagic mass
• "Chicken-wire" calcification on imaging (classic radiological feature)

Microscopy

• Sheets of round to polygonal chondroblasts with distinct cell borders and "coffee bean" (bilobed) nuclei with a longitudinal groove
• Scattered multinucleated osteoclast-type giant cells (reactive)
• Characteristic "chicken-wire" or "pericellular" calcification: fine lace-like calcification around individual chondroblasts
• Variable chondroid matrix (usually scant)

Behaviour

• Locally aggressive; requires curettage + bone grafting
• Rarely metastasises (<2%) - even then called "metastasising chondroblastoma"

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4. Chondromyxoid Fibroma

Epidemiology

• Rarest of common benign cartilage tumours
• Age 10-30 years; M > F

Sites

• Metaphysis of long bones; tibial metaphysis most common (proximal tibia)
• Flat bones (ilium)

Pathogenesis

• GRM1 gene rearrangements (metabotropic glutamate receptor 1)

Macroscopy

• Lobulated, grey-white, firm mass; well-circumscribed, eccentric, often causes cortical expansion

Microscopy

• Stellate to spindled cells in a myxoid/chondroid matrix arranged in lobules
• Hypocellular centres with hypercellular periphery (lobule rim with spindle/round cells)
• Multinucleated giant cells at periphery of lobules
• Minimal nuclear atypia; rare mitoses

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5. Chondrosarcoma (Malignant Chondrogenic Tumour)

Classification

Epidemiology

• Peak age: 30-60 years (adults - key difference from osteosarcoma which peaks in teenagers)
• Male > Female
• Second most common primary malignant bone tumour

Sites

• Central chondrosarcoma: Pelvis > proximal femur > proximal humerus > ribs
• (Contrast with enchondroma which occurs in hands/feet)

Pathogenesis

• De novo (primary): IDH1/IDH2 mutations (75-80%); also CDKN2A deletion, TP53 mutation, loss of RB1
• Secondary (from osteochondroma): Loss of EXT1/EXT2
• Progression: Enchondroma → Grade 1 chondrosarcoma → Grade 2 → Grade 3 via progressive genetic alterations

Grading (Histological)

• Hypercellular cartilage compared to enchondroma
• Mild nuclear atypia; enlarged, hyperchromatic nuclei
• Binucleate cells occasional
• No necrosis; rare mitoses
• Invades Haversian canals (permeation) - key distinguishing feature from enchondroma
• Excellent prognosis (>90% survival)

• Moderate cellularity; myxoid change in matrix
• Moderate nuclear atypia; mitoses present
• Areas of necrosis
• ~60-70% survival

• Highly cellular; marked nuclear pleomorphism
• Numerous mitoses; extensive necrosis
• Spindle cell areas
• ~30% survival

Macroscopy (Conventional)

• Large, lobulated, blue-white-grey glistening mass with gelatinous areas (myxoid change)
• "Rings and arcs" calcification within lobules (key radiological sign)
• Cortical destruction and soft tissue extension in higher grades

Microscopy

• Lobules of cartilage matrix with chondrocytes in lacunae
• Grade-dependent atypia (as above)
• Permeation of host bone trabeculae and Haversian canals (most reliable feature distinguishing chondrosarcoma from enchondroma)
• Myxoid change: loosening of matrix, stellate cells "floating"
• Dedifferentiated: juxtaposition of low-grade cartilage and high-grade spindle cell sarcoma (abrupt interface)

Immunohistochemistry

• S100 protein: positive (marker of chondrogenic differentiation)
• SOX9: positive (transcription factor for chondrogenesis)
• IDH1 R132H mutant-specific antibody: positive in IDH-mutant cases

Molecular Features

• IDH1/IDH2 mutations: Present in enchondroma and Grades 1-3; useful diagnostic marker; therapeutic target (ivosidenib for IDH1)
• CDKN2A deletion: Progression marker
• HEY1-NCOA2 fusion: Diagnostic of mesenchymal chondrosarcoma
• COL2A1 mutations: In ~20%

Treatment and Prognosis

• Surgery is primary treatment (resistant to radiation and conventional chemotherapy)
• Grade 1: Wide local excision (intralesional curettage for accessible lesions)
• Grade 2/3: Wide resection; 5-year survival 60-70% (Grade 2), 30% (Grade 3)
• Dedifferentiated: Very poor prognosis; <10% at 5 years
• Mesenchymal chondrosarcoma: Chemotherapy may help; ~50% at 10 years

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Q3 (20 Marks)

Classification of Adenomas of the Intestine

A. Classification by Morphology (Macroscopic / Endoscopic)

Paris Endoscopic Classification

1. Pedunculated (Ip): Long stalk
2. Sub-pedunculated (Isp): Short stalk
3. Sessile (Is): Broad base, not elevated >2.5 mm
4. Superficial elevated (IIa): Elevated ≤2.5 mm
5. Flat (IIb): No elevation
6. Slightly depressed (IIc): Slightly depressed
7. Excavated (III): Deep ulceration

B. Classification by Histological Architecture

C. Classification by Degree of Dysplasia

• Low-grade dysplasia (LGD): Nuclear stratification confined to lower half; mild atypia
• High-grade dysplasia (HGD): Nuclear stratification reaches luminal surface; marked atypia, complex glandular architecture, cribriform pattern, no invasion through basement membrane
• Intramucosal carcinoma: Invasion into lamina propria (no lymphatic access)

D. Special Syndromes / Polyposis

Familial Adenomatous Polyposis (FAP)

• 100 tubular adenomas throughout colon; APC gene (5q21) mutation

• Autosomal dominant
• 100% cancer risk if untreated; prophylactic colectomy required
• Gardner syndrome: FAP + osteomas + desmoid tumours + epidermoid cysts
• Turcot syndrome: FAP/HNPCC + CNS tumours

Hereditary Non-Polyposis Colorectal Cancer (HNPCC / Lynch Syndrome)

• Mismatch repair gene mutations (MLH1, MSH2, MSH6, PMS2)
• <100 adenomas; right-sided predominance
• Microsatellite instability (MSI-H)

MUTYH-Associated Polyposis (MAP)

• Autosomal recessive; base excision repair defect
• 10-100 adenomas

Serrated Polyposis Syndrome

• 5 serrated polyps proximal to sigmoid, OR >20 anywhere

Peutz-Jeghers Syndrome

• Hamartomatous polyps + mucocutaneous pigmentation; STK11 mutation
• Elevated risk of GI and extra-GI carcinomas

Juvenile Polyposis Syndrome

• Multiple juvenile (retention) polyps; SMAD4 or BMPR1A mutations; elevated cancer risk

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Etiopathogenesis of Colon Carcinoma

Epidemiology

• 3rd most common cancer worldwide; 2nd leading cause of cancer death
• Male > Female (slight); Higher in Western countries (dietary habits)
• Risk increases after age 50

Molecular Pathways to Colon Cancer

Pathway 1: Chromosomal Instability (CIN) Pathway - 85% of sporadic cases

• APC normally promotes degradation of β-catenin
• Mutant APC → β-catenin accumulates → enters nucleus → activates TCF/LEF transcription factors → target genes: MYC (proliferation), cyclin D1 (cell cycle), MMP-7 (invasion)
• APC also regulates microtubule spindle function - explains chromosomal instability (aneuploidy)

Pathway 2: Microsatellite Instability (MSI) Pathway - 15% of sporadic cases; Lynch syndrome

• Loss of DNA mismatch repair (MMR) genes: MLH1 (promoter methylation - sporadic), MSH2, MSH6, PMS2 (germline mutation - Lynch)
• MMR failure → accumulation of mutations in microsatellite repeat regions (unstable repetitive DNA sequences)
• Microsatellites within key tumour suppressor genes (TGFBR2, BAX, IGF2R) undergo frameshift mutations → inactivation
• Results in hypermutation (hypermutator phenotype)

• Right-sided (proximal) tumours predominance
• Medullary / mucinous histology
• Heavy lymphocytic infiltrate ("Crohn's-like" reaction)
• Better prognosis despite higher grade
• Responds to immune checkpoint inhibitors (pembrolizumab)

Pathway 3: CpG Island Methylator Phenotype (CIMP) / Serrated Pathway

• Promoter hypermethylation of tumour suppressor genes (MLH1, CDKN2A)
• BRAF V600E mutation is the initiating event (vs KRAS in CIN)
• Serrated polyp → Sessile serrated lesion (SSL) → Carcinoma
• MLH1 methylation → MSI-H in BRAF-mutated serrated carcinomas (BRAF-mutated MSI carcinomas)
• Also KRAS-mutated pathway → MSS serrated carcinomas
• ~20-30% of colon cancers arise via serrated pathway

Risk Factors for Colon Carcinoma

Modifiable

• Diet: High red/processed meat, high fat, low fibre, low calcium, low vitamin D
• Obesity and physical inactivity: Elevate insulin/IGF-1; hyperinsulinaemia promotes colonocyte proliferation
• Alcohol and tobacco smoking
• Inflammatory bowel disease: Chronic UC (pancolitis >10 years); surveillance colonoscopy essential

Non-Modifiable

• Age: Risk doubles every decade after 40
• Hereditary syndromes: FAP (100% risk), Lynch syndrome (60-80% lifetime risk), MAP
• Previous colorectal adenomas: Personal history increases risk 3-5 fold
• Family history: First-degree relative doubles risk
• Type 2 diabetes / metabolic syndrome
• Acromegaly (elevated IGF-1)

Molecular Features Relevant to Treatment

• KRAS/NRAS mutation: Predicts resistance to anti-EGFR therapy (cetuximab, panitumumab)
• BRAF V600E: Poor prognosis; combination BRAF+MEK inhibitor (encorafenib+binimetinib+cetuximab)
• MSI-H: Excellent response to pembrolizumab (checkpoint inhibitor); FDA approved first-line
• HER2 amplification: ~3% of cases; targetable with trastuzumab/lapatinib
• NTRK fusion: Larotrectinib/entrectinib

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Q4 Short Notes

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Q4a: Interpretation of Liver Biopsy with Reference to Infective Lesions

Approach to Infective Liver Biopsy

• Portal inflammation pattern (hepatitis)
• Granulomatous pattern
• Microabscess / suppurative pattern
• Diffuse sinusoidal / reticuloendothelial pattern
• Necrotising / fibrinous pattern

Specific Infective Lesions

• Acute hepatitis (HAV, HBV, HEV): Lobular disarray; hepatocyte swelling/ballooning degeneration; acidophil (Councilman) bodies (apoptotic hepatocytes); lobular inflammation; portal inflammation with lymphocytes; bridging necrosis in severe cases
• Chronic hepatitis B: Interface hepatitis (piecemeal necrosis); portal fibrosis; "ground-glass" hepatocytes (HBsAg in ER - orcein/Victoria blue positive); HBcAg: nuclear "sanded" nuclei (Shikata orcein); correlate with HBV serology
• Chronic hepatitis C: Portal lymphoid aggregates/follicles; mild steatosis (especially genotype 3); mild bile duct damage; mild interface hepatitis; lobular inflammation; progressive fibrosis
• CMV hepatitis: Large intranuclear inclusions with halo ("owl-eye" cells); microabscesses; useful in immunocompromised
• EBV (infectious mononucleosis): Sinusoidal infiltration by atypical lymphocytes ("beads on a string"); hepatitis pattern
• Herpes simplex: Geographic coagulative necrosis without inflammatory response; Cowdry type A intranuclear inclusions; haemorrhagic necrosis in neonates/immunocompromised
• Yellow fever: Mid-zonal necrosis (zone 2); Councilman bodies; Torres bodies (eosinophilic intranuclear inclusions); fat change
• COVID-19: Mild portal inflammation, macrovesicular steatosis, fibrin thrombi in sinusoids

• Pyogenic liver abscess (E. coli, Klebsiella, Streptococcus, Staphylococcus, anaerobes): Zones of coagulative necrosis surrounded by neutrophilic inflammation, granulation tissue, and fibrous capsule; Gram stain for organisms
• Tuberculosis: Epithelioid granulomas with or without central caseous necrosis; Langhans giant cells; ZN stain / auramine-rhodamine for AFB; culture; GeneXpert; typically hepatic involvement in miliary TB (portal/lobular non-caseating granulomas also seen)
• Brucellosis: Small, non-caseating granulomas in portal tracts; fibrin ring granulomas (rare)
• Typhoid (Salmonella typhi): Typhoid nodules - small collections of macrophages (Kupffer cell hyperplasia); no true granuloma formation; relative lymphopenia

• Schistosomiasis: Schistosome eggs in portal tracts surrounded by granulomas; progressive periportal fibrosis ("pipestem fibrosis" / Symmers fibrosis); PAS stain highlights egg wall
• Hydatid disease (Echinococcus): Cyst wall composed of outer laminated eosinophilic acellular membrane (chitinous) + inner germinal layer (nucleated cells); scolices with hooklets in cyst fluid; compressed liver parenchyma; fibrous host reaction
• Visceral leishmaniasis (Kala-azar): Kupffer cell hyperplasia with engulfed amastigotes (Leishman-Donovan bodies); Giemsa stain
• Malaria: Kupffer cell hyperplasia; haemozoin (malaria pigment) - brown-black granular deposits in Kupffer cells; minimal hepatocyte damage
• Amoebiasis: Amoebic liver abscess: "anchovy paste" (lytic necrosis, no neutrophils); trophozoites with engulfed erythrocytes visible at edge of abscess (PAS positive); fibrous capsule

• Candida: Microabscesses; pseudohyphae + budding yeasts on PAS/GMS
• Histoplasma: Intracellular organisms in macrophages; granulomas; GMS/PAS stain
• Aspergillus: Hyphae with acute angle branching, septate; angioinvasion

• ZN / Auramine-rhodamine: AFB (mycobacteria)
• PAS-D (diastase): Fungi, glycogen
• GMS (Grocott): Fungi
• Giemsa: Leishmania, malaria
• Warthin-Starry: Bartonella, spirochaetes
• Shikata orcein / Victoria blue: HBsAg
• Immunohistochemistry: HBcAg, CMV, HSV, EBV (EBER in situ hybridisation)

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Q4b: Role of Electron Microscopy and Immunofluorescence in Diagnosis of Glomerular Lesions

Immunofluorescence (IF)

• Membranous nephropathy: Granular IgG (and IgG4 subclass) + C3, subepithelial, peripheral capillary loop pattern
• IgA nephropathy: Dominant/co-dominant mesangial IgA; IgG and C3 variable
• Anti-GBM disease (Goodpasture): Linear IgG (and C3) along entire GBM
• Lupus nephritis (Class IV): Full-house; IgG, IgA, IgM, C3, C1q all positive (C1q is characteristically strong)
• ANCA vasculitis: Pauci-immune (no Ig deposits despite severe injury)
• C3 glomerulopathy: C3 dominant without Ig (distinguishes from MPGN type I)
• Amyloidosis: Congo red fluorescence under polarised light (apple-green birefringence); IF with anti-amyloid A or anti-kappa/lambda for typing

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Electron Microscopy (EM)

• Minimal change disease (MCD): Complete (global, >90%) foot process effacement with no deposits; normal GBM; EM is essential for diagnosis since LM is near-normal
• Focal segmental glomerulosclerosis (FSGS): Diffuse (not focal) foot process effacement; microvillous transformation; no deposits
• Membranous nephropathy: Also shows foot process effacement + subepithelial deposits

• Thin basement membrane disease: GBM uniformly thin (<200 nm; normal >300 nm) - benign familial haematuria
• Alport syndrome: GBM irregular thickening with lamellation ("basket-weave" appearance) and thinning; collagen IV α3α4α5 network disruption

• Amyloidosis: Randomly arranged, non-branching fibrils, 8-12 nm diameter; in mesangium and GBM
• Fibrillary GN: Randomly arranged fibrils, 16-24 nm diameter; IgG and C3 deposits
• Immunotactoid GN: Organised microtubular structures, 30-50 nm diameter, often parallel arrangement

• HIV-associated nephropathy (HIVAN): Tubuloreticular inclusions (TRI) in endothelial cells (induced by interferon); also seen in lupus nephritis
• Polyomavirus (BK) nephropathy: Intranuclear viral particles in tubular cells; EM diagnostic

• Cryoglobulinaemic GN: Subendothelial "fingerprint" deposits (curved parallel fibrils with periodicity)
• Lupus: Fingerprint/tubuloreticular deposits (pathognomonic for lupus vs other MPGN patterns) + TRI

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Q4c: Paris System for Reporting Urinary Cytology

Rationale

• Urine cytology is excellent at detecting HGUC (sensitivity ~80%) but poor for low-grade lesions (sensitivity ~30%)
• Previous inconsistent terminology led to management ambiguity
• TPS creates a standardised, reproducible reporting system analogous to Bethesda (thyroid/cervical)

Specimen Adequacy

• Adequate: >10 well-preserved urothelial cells per 10 HPF (for voided urine)
• Instrumented specimens (catheter, barbotage, wash): Generally considered adequate regardless of cellularity
• Unsatisfactory: <10 cells or heavy obscuring blood/inflammation

Paris System Diagnostic Categories

Cytomorphological Criteria

• Superficial (umbrella) cells: large, multinucleated, abundant cytoplasm
• Intermediate urothelial cells: moderate N:C ratio
• No nuclear hyperchromasia or irregular nuclear contour

1. High nuclear-to-cytoplasmic (N:C) ratio: N:C ≥0.7
2. Hyperchromasia: Dark, irregular nuclear chromatin
3. Irregular nuclear contours: Thickened, irregular nuclear membrane
4. Any degree of nuclear abnormality: Mitoses, prominent nucleoli (secondary criteria)

• Cells with some features of HGUC but insufficient for definitive diagnosis
• N:C ratio 0.5-0.7, mild hyperchromasia
• Often reactive, instrumentation-related, or suspicious

• Papillary clusters (papillary urothelial neoplasm of low malignant potential / low-grade papillary urothelial carcinoma)
• Increased cellularity, papillary architecture
• Mild nuclear enlargement and crowding
• Reported as LGUN rather than HGUC - indicates presence of a low-grade papillary lesion

Ancillary Testing

• FISH (UroVysion): Detects gains of chromosomes 3, 7, 17 and loss of 9p21 (CDKN2A); improves sensitivity for HGUC detection; especially useful in AUC category
• Immunocytochemistry: p53, CK20 overexpression, CD44 loss in HGUC
• NMP22 (Nuclear Matrix Protein 22): Quantitative marker; elevated in bladder cancer

Clinical Applications

• Surveillance of known urothelial carcinoma (post-resection)
• Haematuria workup
• Monitoring patients with risk factors (smoking, occupational exposure, cyclophosphamide)
• Diagnosis of carcinoma in situ (CIS): flat HGUC not visible endoscopically

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Q4d: Molecular Profiling of Breast Cancers and Prognosis

Intrinsic Molecular Subtypes (Perou et al., 2000; Sorlie et al., 2003)

Surrogate IHC Classification (Clinical Approximation)

• Since gene expression profiling is not universally available, IHC surrogates are used:
  • ER (Allred score or H-score; ≥1% nuclear staining = positive)
  • PR (≥1% nuclear staining)
  • HER2 (FISH/ISH for amplification; IHC 3+ = positive; 2+ requires FISH)
  • Ki67 (proliferation index; cutoff varies by guideline: 14-20%)

Multigene Assays for Prognostication

• 21-gene recurrence score (RS) assay
• Validated in ER+, HER2-, node-negative (and node-positive) breast cancer
• RS 0-17: Low risk; endocrine therapy alone (TAILORx trial)
• RS 18-30: Intermediate risk; endocrine ± chemo (age/menopausal status guides)
• RS ≥31: High risk; chemo + endocrine therapy
• Genes measured: proliferation (Ki67, STK15, Survivin, CCNB1, MYBL2), invasion (MMP11, CTSL2), HER2 (GRB7, HER2), oestrogen (ER, PR, BCL2, SCUBE2), CD68

• 70-gene expression signature (Amsterdam signature)
• MINDACT trial: 46% of patients classified as high clinical / low genomic risk could safely forgo chemotherapy
• Result: Low genomic risk vs High genomic risk
• FDA-cleared; requires fresh/frozen tissue (now FFPE version available)

• 50-gene assay classifying tumours into Luminal A, Luminal B, HER2-enriched, Basal-like subtypes
• Also provides Risk of Recurrence (ROR) score
• Validated in postmenopausal ER+ patients for distant recurrence

• 12-gene assay + tumour size + nodal status = EPclin score
• Predicts late distant recurrence (>5 years)

• Predicts benefit from extended endocrine therapy (>5 years)
• HOXB13:IL17BR ratio + proliferation genes

Key Molecular Alterations and Their Prognostic/Predictive Significance

TNBC Subtyping (Lehmann Classification)

1. BL1 (Basal-like 1): Cell cycle/DNA damage response genes; responds to platinum/PARP inhibitors
2. BL2 (Basal-like 2): EGFR signalling; growth factor receptor pathways
3. M (Mesenchymal): EMT, stemness; PI3K/mTOR pathway
4. LAR (Luminal androgen receptor): AR expression; responds to androgen receptor antagonists (enzalutamide)

• Immunotherapy (pembrolizumab) is approved for PD-L1+ TNBC (CPS ≥10) and high-risk early TNBC

Tumour Mutational Burden (TMB) and MSI

• High TMB (≥10 mut/Mb): Predicts response to pembrolizumab (tumour-agnostic FDA approval)
• MSI-H: Rare in breast cancer (~1-2%); immunotherapy responsive
• NTRK fusions: Rare (~0.5%); respond to larotrectinib/entrectinib

Prognostic Summary

• Best prognosis: Luminal A (ER+, PR high, HER2-, low Ki67) - 10-year survival >90%
• Intermediate: Luminal B
• HER2-enriched: Dramatically improved with targeted therapy; pCR to neoadjuvant anti-HER2 + chemo = excellent long-term outcome
• TNBC/Basal: Most aggressive; 5-year survival ~70-75% for early disease but drops sharply in metastatic; subset of BRCA-mutated and PD-L1+ patients benefit from targeted/immunotherapy
• Late recurrence is characteristic of ER+ tumours (can recur >10-20 years later); TNBC and HER2+ recurrences occur early (within 5 years)

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