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SAQ ANSWERS (5 Marks Each)

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SAQ 1 - Define Neoplasia; Discuss Nomenclature and Classification of Tumours

Definition of Neoplasia

1. Parenchyma - proliferating neoplastic cells
2. Stroma - supporting connective tissue and blood vessels

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Nomenclature

• Malignant tumours of epithelial origin = Carcinoma (e.g., Adenocarcinoma, Squamous cell carcinoma)
• Malignant tumours of mesenchymal origin = Sarcoma (e.g., Fibrosarcoma, Osteosarcoma)
• Malignant tumours of lymphoid/haematopoietic origin = Lymphoma, Leukaemia
• Tumours containing cells from all three germ layers = Teratoma (benign: mature teratoma; malignant: immature teratoma)

• Hepatoma (actually malignant = hepatocellular carcinoma)
• Mesothelioma (malignant)
• Melanoma (malignant)
• Seminoma (malignant)

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Classification of Tumours

1. Benign
2. Malignant
3. Borderline (intermediate)

1. Epithelial tumours - carcinomas
2. Mesenchymal tumours - sarcomas
3. Mixed tumours - contain both epithelial and mesenchymal elements (e.g., fibroadenoma of breast)
4. Compound tumours - Teratomas (all three germ layers)
5. Tumours of haematopoietic/lymphoid tissue - Lymphomas, Leukaemias

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SAQ 2 - Staging and Grading of Cancer and Its Significance

GRADING

• Higher grade = more aggressive behaviour
• Predicts prognosis (Grade III-IV = poor prognosis)
• Guides chemotherapy decisions
• Example: Gleason grading in prostate cancer

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STAGING

• T (Tumour): T1-T4 based on size and local invasion
• N (Nodes): N0 = no nodal involvement; N1-N3 = regional node spread
• M (Metastasis): M0 = no distant metastasis; M1 = distant metastasis

• Most important indicator of prognosis (Stage I = best prognosis; Stage IV = worst)
• Determines treatment plan (surgery vs. chemotherapy vs. radiation)
• Enables uniform communication between clinicians
• Staging is more clinically important than grading for treatment decisions

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SAQ 3 - Describe 4 Modes of Tumour Spread with Examples

Definition

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ROUTES OF TUMOUR SPREAD

• Tumour cells invade adjacent tissues directly.
• Malignant cells secrete proteases (matrix metalloproteinases - MMPs) and collagenases that degrade extracellular matrix.
• Example: Carcinoma of cervix invades the bladder and rectum; Breast cancer invades the overlying skin.

• Most common route for carcinomas (epithelial tumours).
• Tumour cells enter lymphatics and are carried to regional lymph nodes.
• Lymph node enlargement may be due to:
  • Tumour metastasis (malignant)
  • Reactive hyperplasia (benign)
• "Sentinel lymph node" = first draining lymph node; biopsy used to assess spread.
• Example: Carcinoma of breast - spreads to axillary lymph nodes. Carcinoma of stomach - Virchow's node (left supraclavicular).

• Typical route for sarcomas (mesenchymal tumours), but also seen in carcinomas.
• Tumour cells enter veins (veins preferentially over arteries due to thinner walls).
• Venous drainage determines site of metastasis:
  • Portal vein drainage → Liver metastases (e.g., GI tract carcinomas)
  • Systemic veins → Lung metastases
  • Vertebral venous plexus (Batson's plexus) → Vertebral column (e.g., prostate, thyroid)
• Example: Renal cell carcinoma → lungs and bone; Hepatocellular carcinoma → lungs.

• Tumour cells shed into body cavities and implant on serosal surfaces.
• Most common in peritoneal cavity.
• Example: Carcinoma of ovary seeds the peritoneum, omentum, and bowel serosa producing "studded" pattern. Pseudomyxoma peritonei from mucinous carcinoma of appendix or ovary. Krukenberg tumour = bilateral ovarian metastases from gastric carcinoma (via transperitoneal spread).

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SAQ 4 - Define Neoplasia; Differences Between Benign and Malignant Tumours

DIFFERENCES BETWEEN BENIGN AND MALIGNANT TUMOURS

Key Point from Robbins: Metastasis is the single most reliable indicator of malignancy. The ability to metastasize distinguishes benign from malignant tumours.

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SAQ 5 - Oncogenes and Antioncogenes

ONCOGENES

1. Point mutation - e.g., RAS mutation (most common; found in ~30% of all human cancers) - single nucleotide change causing constitutively active RAS protein
2. Gene amplification - multiple copies of oncogene; e.g., N-MYC amplification in neuroblastoma; HER2/NEU in breast cancer
3. Chromosomal translocation / gene rearrangement - e.g., BCR-ABL fusion (Philadelphia chromosome t(9;22)) in CML; C-MYC overexpression in Burkitt lymphoma t(8;14)

• Growth factors: PDGF-B (overexpression)
• Growth factor receptors: HER2/NEU (receptor tyrosine kinase; overexpressed in breast cancer)
• Signal transducers: RAS (GTP-binding proteins)
• Nuclear transcription factors: MYC
• Cell cycle regulators: Cyclin D1 overexpression

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ANTIONCOGENES (Tumour Suppressor Genes)

• RB gene (retinoblastoma): controls cell cycle at G1/S checkpoint
• TP53: "guardian of the genome"
• APC: controls Wnt signalling; lost in colon cancer
• BRCA1/BRCA2: DNA repair; lost in familial breast/ovarian cancer

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SAQ 6 - Tumour Suppressor Genes (in detail)

Definition

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RB GENE (Retinoblastoma Gene)

• Located on chromosome 13q14
• The RB protein (pRb) is the "Governor of the Cell Cycle"
• In its hypophosphorylated (active) form, pRb binds and inhibits E2F transcription factors, preventing S-phase entry
• When cells receive growth signals: Cyclin D - CDK4/6 complex phosphorylates pRb → pRb releases E2F → cell enters S phase and divides
• When RB is mutated/lost: E2F is constitutively active → uncontrolled cell cycle progression
• Retinoblastoma: Loss of both RB alleles
  • Familial form: one germline mutation (inherited) + one somatic hit
  • Sporadic form: two somatic mutations
• Also implicated in: osteosarcoma, breast, lung, colon cancers

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TP53 GENE (p53 - "Guardian of the Genome")

• Located on chromosome 17p13
• Most commonly mutated gene in human cancers (~50% of all cancers have TP53 mutations)
• Normal p53 functions as a transcription factor activated by DNA damage, hypoxia, oncogene activation

1. G1 arrest - p53 activates CDKN1A (p21), which inhibits Cyclin-CDK complexes → cell pauses for repair
2. DNA repair - induces DNA repair genes
3. Apoptosis - if repair fails, p53 activates BAX and other proapoptotic genes
4. Senescence - permanent cell cycle arrest in severely damaged cells

• In normal cells: MDM2 protein constantly degrades p53 (keeping levels low)
• After DNA damage: kinases (ATM, Chk2) phosphorylate p53 → breaks MDM2-p53 interaction → p53 accumulates → activates downstream genes

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SAQ 7 - P53 Oncogene and Retinoblastoma Gene

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LAQ ANSWERS (10 Marks Each)

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LAQ 1 - Define Metastasis; Discuss Mechanism and Routes of Tumour Metastasis

DEFINITION

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MECHANISM OF METASTASIS

1. Detachment - loosening of cell-cell contacts by downregulation of E-cadherin (an adhesion molecule)
2. Attachment - tumour cells attach to ECM components (laminin, fibronectin, collagen) via integrins
3. Degradation of ECM - secretion of proteolytic enzymes:
   • Matrix metalloproteinases (MMPs) - collagenases, gelatinases
   • Serine proteases (plasminogen activator)
   • These enzymes also release growth factors (VEGF, FGF) stored in ECM
4. Migration - tumour cells migrate through degraded matrix via cytoskeletal reorganisation (pseudopodia formation)

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• Tumour cells enter lymphatics or blood vessels
• Small venules and lymphatics are more easily penetrable than arteries
• Tumour cells may travel as single cells or as tumour cell emboli (often mixed with platelets - which protect them from immune attack)

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• Most tumour cells die in the circulation due to mechanical trauma and immune destruction (NK cells)
• Cells that survive form microemboli with fibrin-platelet aggregates - this protects them

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• Tumour cells adhere to endothelium at target site (again mediated by cell-surface lectins and integrins)
• Cross the basement membrane using proteases
• Enter target tissue

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• Not all cells that arrive at a distant organ can form metastases
• The microenvironment of the target organ must support tumour growth ("seed and soil" hypothesis - Paget, 1889)
• Tumour cells produce autocrine growth factors and co-opt resident stromal cells
• Angiogenesis at the new site is stimulated by VEGF

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ROUTES OF METASTASIS

• Most common for carcinomas
• Tumour spreads to regional lymph nodes first, then further
• Sentinel node biopsy concept
• Examples: Breast ca → axillary nodes; Lung ca → hilar and mediastinal nodes; Stomach ca → Virchow's node (left supraclavicular - Troisier's sign)

• Most common for sarcomas
• Via veins (thin-walled, less pressure than arteries)
• Portal vein drainage → liver metastases (colon, stomach, pancreas)
• Systemic veins → lungs
• Vertebral (Batson's) plexus → vertebrae and brain (prostate, thyroid, kidney)
• Lungs are most common site of blood-borne metastases; liver is second

• Spread through peritoneal, pleural, pericardial cavities
• Tumour cells shed from the primary and implant on serosal surfaces
• Example: Ovarian carcinoma → peritoneum and omentum ("omental cake")
• Krukenberg tumour: bilateral ovarian metastases from gastric carcinoma (via transcoelomic spread)

• Invasion of adjacent structures
• Example: Carcinoma cervix invades bladder and rectum; Pancreatic cancer invades duodenum

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• Certain tumours preferentially metastasize to specific organs beyond what anatomy predicts
• Prostate carcinoma → bone (especially lumbar vertebrae)
• Breast carcinoma → bone, liver, lung, brain, adrenal
• Bronchogenic carcinoma → adrenal, brain, liver, bone
• Colon carcinoma → liver

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LAQ 2 - Define Neoplasia; Molecular Basis of Cancers; Note on Oncogenes and Their Mode of Activation

DEFINITION OF NEOPLASIA

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MOLECULAR BASIS OF CANCER

A. Proto-Oncogenes → Oncogenes (Gain-of-function mutations)

1. Point Mutations
   • Single nucleotide change → altered protein function
   • Most important example: RAS mutations in ~30% of all cancers
   • Normal RAS: GTPase activity turns off growth signals after binding GTP
   • Mutated RAS: GTPase activity lost → permanently active (stuck in GTP-bound state) → constitutive proliferative signalling
2. Gene Amplification
   • Multiple copies of oncogene → excessive protein
   • N-MYC: amplified in neuroblastoma (poor prognosis)
   • HER2/NEU: amplified in breast cancer (targeted by trastuzumab/Herceptin)
   • MYC: amplified in lung, colon, breast
3. Chromosomal Translocation / Rearrangement
   • Gene moved near powerful promoter or creates fusion gene
   • t(9;22) BCR-ABL (Philadelphia chromosome) in CML → constitutively active tyrosine kinase → targeted by imatinib (Gleevec)
   • t(8;14) in Burkitt lymphoma: MYC placed under Ig heavy chain promoter → MYC overexpression

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B. Tumour Suppressor Genes (Loss-of-function)

• RB gene - loss of G1/S checkpoint control (Retinoblastoma)
• TP53 - loss of DNA damage response; mutated in ~50% of cancers
• APC - loss leads to activation of Wnt/β-catenin pathway; lost in familial adenomatous polyposis and sporadic colon cancer
• BRCA1/BRCA2 - loss of DNA repair; familial breast and ovarian cancer

• Both alleles of a tumour suppressor gene must be inactivated
• Hereditary cancers: 1st hit = germline mutation (inherited); 2nd hit = somatic mutation
• Sporadic cancers: both hits are somatic

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C. Genes Regulating Apoptosis

• BCL2 overexpression prevents apoptosis; found in follicular lymphoma via t(14;18)
• Loss of FAS/FASL pathway
• Mutations inactivating BAX

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D. Genes Regulating DNA Repair (Mutator Genes / Caretaker Genes)

• Loss of DNA mismatch repair (MMR) genes → microsatellite instability → Lynch syndrome (HNPCC)
• Loss of nucleotide excision repair (NER) → Xeroderma pigmentosum → skin cancer
• Loss of BRCA1/2 → impaired homologous recombination → breast/ovarian cancer

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E. Genes Regulating Telomeres

• Normal cells: telomeres shorten with each division → cellular senescence
• Cancer cells: telomerase (reverse transcriptase) is reactivated → restores telomere length → limitless replicative potential (immortality)

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F. Epigenetic Changes

• Promoter hypermethylation silences tumour suppressor genes (e.g., silencing of RB, CDKN2A/p16 by methylation)
• Histone deacetylation → gene silencing
• MicroRNAs (miRNAs): tumour suppressor miRNAs are lost; oncogenic miRNAs are overexpressed (oncomiRs)

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Hallmarks of Cancer (Hanahan & Weinberg)

1. Self-sufficiency in growth signals (oncogenes)
2. Insensitivity to growth-inhibitory signals (tumour suppressor loss)
3. Evasion of apoptosis
4. Limitless replicative potential (telomerase activation)
5. Sustained angiogenesis (VEGF)
6. Invasion and metastasis
7. Reprogramming of energy metabolism (Warburg effect)
8. Evasion of immune destruction

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LAQ 3 - Carcinogenesis: Physical, Chemical, Biological and Molecular Basis

CARCINOGENESIS

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A. CHEMICAL CARCINOGENESIS

• Alkylating agents: cyclophosphamide (also used as chemotherapy, paradoxically causes secondary cancer)
• Acylating agents: β-propiolactone
• Nitrosamides

• Polycyclic aromatic hydrocarbons (PAH): benzopyrene in tobacco smoke → lung cancer
• Aromatic amines & azo dyes: β-naphthylamine → bladder cancer (industrial exposure)
• Aflatoxin B1: produced by Aspergillus flavus on stored grains → hepatocellular carcinoma (forms DNA adducts at codon 249 of TP53)
• Nitrosamines: in smoked foods → gastric cancer
• Vinyl chloride → hepatic angiosarcoma

• Two phases:
  1. Initiation: Irreversible DNA damage (mutation); initiated cells are not yet neoplastic
  2. Promotion: Prolonged exposure to promoting agents (e.g., phorbol esters, saccharin) → clonal expansion of initiated cells. Reversible if promoter removed early.

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B. RADIATION CARCINOGENESIS

1. Ultraviolet radiation (UV):
   • UVB (280-320 nm) is most carcinogenic
   • Causes pyrimidine dimers (cyclobutane dimers) in DNA
   • Normally repaired by nucleotide excision repair (NER)
   • Loss of NER → Xeroderma pigmentosum → squamous cell carcinoma, basal cell carcinoma, melanoma
   • Ozone layer depletion → increased UV-related skin cancers
2. Ionising Radiation (X-rays, gamma rays, particles):
   • Causes double-strand DNA breaks and generation of free radicals
   • Survivors of Hiroshima/Nagasaki: increased incidence of leukaemia (especially CML, AML), thyroid cancer, breast cancer
   • Medical radiation: radiologists historically had increased cancer risk
   • Thyroid cancer in children exposed to radioactive iodine (Chernobyl)
   • Radon gas inhalation → lung cancer in miners

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C. BIOLOGICAL CARCINOGENESIS (Viral and Microbial Oncogenesis)

• HTLV-1 (Human T-cell Leukaemia Virus type 1) → Adult T-cell Leukaemia/Lymphoma
  • HTLV-1 encodes TAX protein → activates NF-κB and cyclins → T-cell proliferation

• HPV 16/18 viral proteins E6 and E7 are oncoproteins
• E6 binds and degrades p53 (inactivates tumour suppressor)
• E7 binds and inactivates pRb (releases E2F → uncontrolled cell cycle)

• Causes chronic gastritis → gastric adenocarcinoma and gastric MALT lymphoma
• Mechanism: chronic inflammation → free radical generation → DNA damage; also CagA virulence factor activates signalling pathways

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D. MOLECULAR BASIS OF CARCINOGENESIS

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LAQ 4 - Multistep Carcinogenesis; Role of Tumour Suppressor Genes

MULTISTEP CARCINOGENESIS

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Classic Model: Colorectal Carcinogenesis (Vogelstein's Model)

Normal epithelium
       ↓ Loss of APC (tumour suppressor; chromosome 5q)
Early adenoma (small polyp)
       ↓ KRAS activation (point mutation)
Intermediate adenoma
       ↓ Loss of SMAD2/SMAD4 (chromosome 18q)
Late adenoma (large polyp, dysplastic)
       ↓ Loss of TP53 (chromosome 17p)
Carcinoma-in-situ
       ↓ Additional mutations
Invasive carcinoma → Metastasis

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TUMOUR PROGRESSION

• As tumours evolve, genetically distinct subclones emerge
• Clonal evolution: subclones with additional mutations that confer greater growth advantage outcompete others
• Results in tumours that are increasingly:
  • Aggressive
  • Drug-resistant
  • Capable of invasion and metastasis
• This is why tumours are heterogeneous and why treatment resistance develops

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ROLE OF TUMOUR SUPPRESSOR GENES IN CARCINOGENESIS

1. RB Gene - Cell Cycle Regulation

• Normal: pRb (hypophosphorylated) binds and sequesters E2F transcription factors → cells stay in G0/G1
• Cell receives growth signals: Cyclin D-CDK4/6 → phosphorylates pRb → releases E2F → S phase entry
• Mitogenic stimuli keep CDK active: normal growth
• Loss of RB: E2F is always free → uncontrolled G1→S transition → uncontrolled proliferation
• Clinical: Retinoblastoma (childhood eye tumour); osteosarcoma; lung, breast, bladder cancers

• Hereditary retinoblastoma: one mutant RB allele inherited; second hit is somatic → bilateral, early onset
• Sporadic retinoblastoma: both hits are somatic → unilateral, late onset

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2. TP53 - Guardian of the Genome

• Activated by: DNA damage, oncogene activation, hypoxia
• Actions: G1 arrest (via p21), DNA repair, apoptosis (via BAX), senescence
• In cancer: loss of p53 = DNA damage goes unrepaired → accumulation of mutations → malignant transformation
• Li-Fraumeni syndrome: germline TP53 mutation → multiple cancers (sarcomas, breast, brain) at young age

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3. APC Gene (Adenomatous Polyposis Coli)

• Located on chromosome 5q21
• APC protein normally inhibits the Wnt signalling pathway by promoting degradation of β-catenin
• Loss of APC → β-catenin accumulates in nucleus → activates target genes (MYC, cyclin D1) → cell proliferation
• Familial Adenomatous Polyposis (FAP): germline APC mutation → hundreds to thousands of colonic polyps → inevitable colorectal carcinoma by age 40

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4. BRCA1 / BRCA2

• DNA repair genes (homologous recombination)
• Loss → DNA damage unrepaired → accumulation of mutations
• BRCA1/2 mutations: familial breast and ovarian cancers
• 55-65% lifetime risk of breast cancer with BRCA1 mutation

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5. CDKN2A (p16/INK4a)

• Encodes both p16 (INK4a) and p14 (ARF) from the same locus
• p16 inhibits CDK4/6 → prevents pRb phosphorylation → blocks G1/S
• p14 stabilises p53 by inhibiting MDM2
• Loss of CDKN2A = loss of both RB pathway and p53 pathway brakes simultaneously
• Frequently deleted in melanoma, pancreatic cancer

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Summary Table - Key Tumour Suppressor Genes

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