Neoplasia: A Comprehensive Review

Based on Robbins & Cotran Pathologic Basis of Disease

7. What is Carcinogenesis? Causes: Chemical, Viral, and Radiation

1. Initiation - exposure to a carcinogenic agent causes permanent, heritable DNA damage (mutation). Initiated cells are not yet tumors.
2. Promotion - promoters (not themselves mutagenic) cause clonal expansion of initiated cells. Promotion is reversible in early stages.
3. Progression - subclones accumulate additional mutations, eventually yielding a fully malignant, invasive cancer.

A. Chemical Carcinogenesis

• Alkylating agents: β-propiolactone, dimethyl sulfate, diepoxybutane
• Anticancer drugs: cyclophosphamide, chlorambucil, nitrosoureas
• These are weak carcinogens but important because therapeutic use can induce secondary malignancies (especially acute myeloid leukemia)

• Polycyclic aromatic hydrocarbons (PAH): benzo[a]pyrene (from cigarette smoke, soot, grilled meats) - most potent indirect carcinogens. Metabolized by CYP1A1 to reactive epoxides.
• Aromatic amines and azo dyes: 2-naphthylamine (bladder cancer in dye workers), benzidine
• Aflatoxin B1: produced by Aspergillus flavus on peanuts/grains - causes hepatocellular carcinoma, characteristically mutates codon 249 of TP53
• Nitrosamines: formed from dietary nitrites - gastric cancer
• Vinyl chloride: angiosarcoma of liver

B. Radiation Carcinogenesis

Ultraviolet (UV) Radiation

• UV-B (wavelength 280-320 nm) is the main carcinogenic component of sunlight
• Causes pyrimidine dimers (covalent cross-links between adjacent thymine/cytosine residues) in DNA
• Repaired by nucleotide excision repair (NER); deficiency in NER causes xeroderma pigmentosum with >1000-fold increased risk of skin cancer
• Causes squamous cell carcinoma, basal cell carcinoma, and melanoma
• Dose-dependent: cumulative lifetime UV exposure correlates with skin cancer risk

Ionizing Radiation

• X-rays, gamma rays, alpha/beta particles cause double-stranded DNA breaks and chromosomal rearrangements
• Historical evidence: thyroid cancer from radioiodine fallout (Chernobyl, atomic bomb survivors), leukemia in radiologists, lung cancer in uranium miners (radon exposure)
• Increased leukemia risk after diagnostic/therapeutic radiation
• Mechanism: direct ionization of DNA or indirect damage via reactive oxygen species
• Robbins & Cotran, pp. 288-289

C. Microbial (Viral) Carcinogenesis

Oncogenic RNA Viruses (Retroviruses)

• HTLV-1 (Human T-cell Leukemia Virus type 1): causes adult T-cell leukemia/lymphoma
  • Encodes the Tax protein, which activates NF-κB and transcription of growth-promoting genes including IL-2 and IL-2 receptor, creating an autocrine growth loop
  • Tax also inhibits TP53 function and DNA repair

Oncogenic DNA Viruses

• More than 100 HPV subtypes; HPV 16, 18 are high-risk for carcinoma
• Encodes two oncoproteins: E6 and E7
  • E6 binds and promotes ubiquitin-mediated degradation of p53 (TP53)
  • E7 binds and inactivates RB protein, releasing E2F transcription factors and driving S-phase entry
  • E7 also binds p21 and p27, bypassing additional cell cycle checkpoints
• Causes cervical carcinoma, oropharyngeal cancer, penile/anal/vulvar cancers
• In cervical cancer, HPV DNA is integrated into host genome (unlike benign warts where it remains episomal)

• Latently infects B lymphocytes; encodes LMP-1 (latent membrane protein-1), a functional mimic of the CD40 receptor
• LMP-1 activates NF-κB and JAK/STAT pathways, driving B-cell proliferation
• Associated with: Burkitt lymphoma (Africa), diffuse large B-cell lymphoma (immunosuppressed), Hodgkin lymphoma, nasopharyngeal carcinoma
• In Burkitt lymphoma, EBV infection precedes the characteristic t(8;14) translocation (MYC to immunoglobulin locus)

• Major cause of hepatocellular carcinoma (HCC) worldwide
• HBx protein activates transcription of multiple proto-oncogenes
• Chronic infection causes cycles of liver cell destruction/regeneration, promoting accumulation of mutations
• HBV DNA integrates near and disrupts various growth-regulatory genes

• RNA virus (not integrating) - causes HCC primarily through chronic hepatitis, cirrhosis, and sustained regenerative stimulus
• HCV proteins may directly interact with p53 and Wnt/β-catenin pathways

• Associated with gastric adenocarcinoma and MALT lymphoma
• Causes chronic gastritis, stimulating B-cell proliferation via T-cell activation
• H. pylori eradication can cause regression of early MALT lymphomas, confirming the driver role of chronic antigen stimulation
• Robbins & Cotran, pp. 289-292

1. Molecular Basis of Carcinogenesis

Multistep Model - Colorectal Carcinoma as the Prototype

1. Normal epithelium - APC mutation (early event, familial and sporadic)
2. Hyperproliferative epithelium - Hypomethylation of DNA
3. Early adenoma - KRAS mutation
4. Intermediate adenoma - Loss of SMAD2/4 (18q deletion)
5. Late adenoma - TP53 mutation (late event)
6. Carcinoma - Additional mutations in telomerase, PI3K, etc.

Genomic Instability as an Enabling Characteristic

• MMR proteins (MSH2, MLH1, MSH6, PMS2) act as "spell checkers" during DNA replication
• Loss of MMR leads to microsatellite instability (MSI) - expansion or contraction of repetitive sequences throughout the genome
• Germline MMR mutations cause Lynch syndrome (HNPCC) - autosomal dominant, predominantly proximal colon cancers
• Sporadic MSI cancers (15% of colon cancers) usually result from epigenetic silencing of MLH1

• NER repairs UV-induced pyrimidine dimers
• Germline NER gene mutations cause xeroderma pigmentosum - extreme sensitivity to UV and high rates of skin cancer

• Repairs double-strand DNA breaks and cross-links
• Germline BRCA1/BRCA2 mutations impair HR, causing familial breast and ovarian cancer
• ATM mutations cause ataxia-telangiectasia (leukemia/lymphoma risk)
• Bloom syndrome (helicase mutation), Fanconi anemia (>12 genes) - impaired HR and cancer predisposition

Epigenetic Changes in Cancer

• Genome-wide DNA hypomethylation: activates proto-oncogenes and promotes chromosomal instability
• Promoter CpG island hypermethylation: silences tumor suppressor genes (e.g., MLH1 in sporadic MSI cancers, CDKN2A/p16 in many cancers)
• Histone modifications: H3K27 trimethylation by EZH2 silences differentiation genes
• Mutations in chromatin-remodeling enzymes: SWI/SNF complex (ARID1A, SMARCB1) mutations are common drivers in ovarian clear cell, rhabdoid tumors, and others
• Noncoding RNAs: miRNAs regulate hundreds of target mRNAs; oncomiRs (e.g., miR-21) are overexpressed and silence tumor suppressors; tumor suppressor miRNAs (e.g., let-7, miR-34) are frequently silenced in cancer

2. Hallmarks of Cancer

The Eight Hallmarks

• Tumors proliferate without external growth factor stimuli
• Mechanisms: autocrine growth factor loops (gliomas expressing PDGF + PDGFR), gain-of-function mutations in growth factor receptors (EGFR in lung cancer), constitutively active signal transducers (RAS mutations in ~30% of all cancers)
• Proto-oncogene activation is the molecular basis

• Tumor cells fail to respond to growth-inhibitory molecules (TGF-β, contact inhibition)
• Mechanisms: inactivation of RB (retinoblastoma protein), loss of TP53, inactivation of CDK inhibitors (p16/CDKN2A, p21)
• Tumor suppressor gene loss is the molecular basis

• Even in normoxic conditions, cancer cells preferentially use aerobic glycolysis (glucose → lactate) rather than oxidative phosphorylation
• This "Warburg effect" supports rapid biosynthesis of macromolecules (lipids, nucleotides, proteins) needed for cell growth
• HIF-1α (activated by hypoxia or oncogenic signaling) drives expression of glycolytic enzymes
• MYC amplification upregulates glutamine metabolism
• Oncometabolism: IDH1/2 mutations in gliomas and AML produce the oncometabolite 2-hydroxyglutarate (2-HG), which inhibits α-ketoglutarate-dependent enzymes (histone demethylases, TET DNA demethylases), causing a hypermethylation phenotype
• Autophagy: cancer cells can cannibalize their own organelles for energy during nutrient deprivation; becomes a survival mechanism in established tumors

• Tumors resist programmed cell death
• Mechanisms: BCL-2 overexpression (follicular lymphoma - t(14;18)), loss of TP53, overexpression of IAPs (inhibitor of apoptosis proteins), upregulation of survival signaling via PI3K/AKT pathway
• BCL-2 family: BCL-2, BCL-XL are anti-apoptotic; BAX, BAK are pro-apoptotic; cancer tips the balance toward survival

• Normal cells undergo senescence after ~70 doublings (Hayflick limit) due to telomere shortening
• Cancer cells acquire telomerase (TERT) expression, preventing telomere erosion and enabling immortal replication
• Cancer stem cells possess inherent self-renewal capacity
• Two mechanisms for cancer stemness: transformation of a tissue stem cell (e.g., HSC in CML) or acquisition of stemness mutation in a progenitor (e.g., granulocyte progenitor in APL)

• Tumors >1-2 mm cannot grow without a new blood supply
• The angiogenic switch: balance shifts from anti-angiogenic to pro-angiogenic factors
• Key pro-angiogenic factor: VEGF (upregulated by HIF-1α under hypoxia, and by oncogenes like RAS and MYC)
• Anti-angiogenic factors: thrombospondin-1 (upregulated by p53), angiostatin, endostatin
• Loss of p53 removes thrombospondin-1 upregulation, permitting VEGF to dominate
• Therapeutic targeting: bevacizumab (anti-VEGF) now part of oncology armamentarium

• Metastasis causes ~90% of cancer deaths
• Invasion of ECM: requires loss of E-cadherin (epithelial-mesenchymal transition/EMT), degradation of basement membrane by matrix metalloproteinases (MMPs), acquisition of motility by tumor cells
• EMT - tumor cells downregulate E-cadherin (adhesion), upregulate N-cadherin and vimentin (mesenchymal markers), acquire motility and invasive capacity
• Vascular dissemination: circulating tumor cells (CTCs) form multicellular aggregates with platelets for protection; express CD44 and chemokine receptors for tissue homing
• Seed and soil hypothesis (Paget): metastatic cells (seeds) preferentially colonize permissive tissues (soil) - e.g., prostate/breast cancer to bone, lung cancer to adrenals/brain
• Colonization involves: surviving anoikis, evading immune surveillance, adapting to new metabolic microenvironment, escaping from tumor dormancy

• Tumor immune evasion mechanisms:
  • Downregulation of MHC class I on tumor cells (invisible to CTLs)
  • PD-L1/PD-L2 upregulation on tumor cells - engages PD-1 on T cells, inducing T-cell anergy/exhaustion
  • CTLA-4 signaling: tumor regulatory T cells (Tregs) express CTLA-4, competing with CD28 for B7 ligands and suppressing effector T cell activation
  • Production of immunosuppressive cytokines: TGF-β, IL-10
  • Induction of immune tolerance through FasL expression on tumor cells (killing Fas+ T cells)
  • Secretion of IDO (indoleamine 2,3-dioxygenase) - depletes tryptophan needed for T-cell function

Two Enabling Characteristics

1. Genomic Instability: Accelerates acquisition of driver mutations (discussed above)
2. Cancer-Enabling Inflammation: Tumor-associated macrophages (TAMs) and other inflammatory cells supply growth factors, survival signals, pro-angiogenic factors, and matrix-remodeling enzymes that support tumor progression

3. Oncogenes

Mechanisms of Proto-Oncogene Activation

• RAS genes (KRAS, NRAS, HRAS): most commonly mutated oncogenes in human cancer (~30% of all tumors)
• RAS is a GTPase that transmits signals from receptor tyrosine kinases to downstream effectors (RAF-MEK-ERK and PI3K-AKT)
• Oncogenic RAS mutations (codons 12, 13, 61) impair intrinsic GTPase activity - RAS stays locked in the active GTP-bound state
• KRAS mutations are found in >90% of pancreatic cancers, ~50% of colon cancers, ~30% of lung adenocarcinomas

• Multiple copies of oncogene DNA sequences appear as:
  • Double minutes (small extrachromosomal circular DNA pieces)
  • Homogeneous staining regions (HSRs) - inserted amplified DNA creating bands lacking normal light/dark banding pattern
• NMYC: amplified in 25-30% of neuroblastomas; associated with poor prognosis
• ERBB2 (HER2): amplified in ~20% of breast cancers; target of trastuzumab (Herceptin)
• EGFR: amplified in glioblastoma and some NSCLC
• CCND1 (Cyclin D1): amplified in mantle cell lymphoma (also via translocation)

• t(9;22) - Philadelphia chromosome: BCR::ABL1 fusion in CML (and ALL)
  • Creates a constitutively active tyrosine kinase; BCR-ABL activates RAS, PI3K, and JAK/STAT
  • Targeted by imatinib (first targeted tyrosine kinase inhibitor)
• t(8;14): MYC translocation to IgH locus in Burkitt lymphoma - massive MYC overexpression
• t(15;17): PML::RARα fusion in acute promyelocytic leukemia (APL)
  • PML-RARα acts as a dominant-negative transcription repressor blocking myeloid differentiation
  • ATRA (all-trans retinoic acid) binds PML-RARα, relieving repression and enabling differentiation - the first successful differentiation therapy
• EML4::ALK fusion (inv(2p)) in ~5% of lung adenocarcinomas - constitutively active ALK kinase; targeted by crizotinib

• Small deletions can activate oncogenes by juxtaposing them with active promoters
• TAL1 overexpression in T-ALL via promoter juxtaposition (chromosome 1 deletion)

Categories of Oncoproteins (Table 7.5 in Robbins)

MYC - A Master Transcription Factor Oncogene

• MYC (c-Myc) is a transcription factor that forms heterodimers with MAX to activate transcription of hundreds of target genes
• When overexpressed, MYC drives cell proliferation (upregulates cyclins, CDKs), metabolism (upregulates glutamine transporter, glycolytic enzymes), and angiogenesis (VEGF)
• MYC also causes genomic instability and sensitizes cells to apoptosis - hence its combination with BCL-2 overexpression in many lymphomas

Cyclins and CDKs as Oncoproteins

• CDK4/Cyclin D1 complex phosphorylates RB, releasing E2F and allowing S-phase entry
• CDK inhibitors (INK4 family: p16, p15; CIP/KIP family: p21, p27) brake this process
• CCND1 amplification/overexpression constitutively activates CDK4/6 and keeps RB phosphorylated (inactive) permanently
• CDK4/6 inhibitors (palbociclib, ribociclib) exploit this in breast cancer treatment

4. Tumor Suppressor Genes

RB: Governor of Proliferation

• RB protein (pRb) is a "master brake" on the cell cycle
• In its hypophosphorylated (active) state, pRb binds and inhibits E2F transcription factors, blocking transcription of genes needed for S-phase entry
• Growth factors activate Cyclin D/CDK4/6 complexes → phosphorylate RB → RB releases E2F → cell enters S phase

• Germline RB mutation (one allele) + somatic loss of second allele = retinoblastoma (Knudson's two-hit)
• Somatic mutations in both RB alleles occur in: osteosarcoma, breast, lung (SCLC), bladder carcinomas
• Functional inactivation even without RB mutation: cyclin D overexpression (keeps RB phosphorylated), loss of p16/CDKN2A (fails to inhibit CDK4/6), viral oncoproteins (HPV E7 binds and inactivates pRb)

• Familial retinoblastoma: one mutant RB inherited (first hit germline) + one somatic mutation (second hit) → nearly all children develop bilateral, multifocal tumors
• Sporadic retinoblastoma: both hits must be somatic → unilateral, unifocal, later onset

TP53: Guardian of the Genome

• p53 is a transcription factor activated by cellular stresses (DNA damage, hypoxia, oncogene activation)
• In unstressed cells, p53 levels are kept low by MDM2, an E3 ubiquitin ligase that promotes proteasomal degradation of p53
• DNA-damage-induced kinases (ATM, ATR, Chk1/Chk2) phosphorylate p53 at Ser15/Ser20, preventing MDM2 binding → p53 accumulates

1. Transient cell cycle arrest (G1 arrest):
   • p53 transcribes CDKN1A (p21) → p21 inhibits CDK4/D cyclin complexes → RB stays active → G1 block
   • Allows time for DNA repair (p53 also induces GADD45 repair genes)
   • If repair successful: MDM2 re-accumulates, p53 falls, cell cycle resumes
2. Senescence (permanent arrest):
   • Epigenetic changes form heterochromatin at cell cycle gene loci
   • Irreversible, stress-dependent
3. Apoptosis:
   • p53 transcribes pro-apoptotic genes: BAX (promotes cytochrome c release), PUMA, NOXA
   • Triggered when DNA damage is too severe for repair

• Li-Fraumeni syndrome: germline TP53 mutation → multiple cancers (sarcomas, leukemia, brain tumors, breast cancer) at young age
• Somatic mutations: missense mutations in the DNA-binding domain (codons 175, 248, 249, 273 are hotspots)
• Aflatoxin B1 signature: G→T transversion at codon 249
• Viral inactivation: HPV-E6 targets p53 for degradation; HBx may inhibit p53 transcriptional activity
• MDM2 amplification: amplified in ~7% of cancers (especially well-differentiated liposarcoma), keeping p53 degraded even when intact

Other Key Tumor Suppressor Genes

• Normally degrades β-catenin (the effector of Wnt signaling)
• When APC is mutated, β-catenin accumulates, translocates to nucleus, and activates MYC, Cyclin D1, and other proliferative genes
• Germline APC mutations cause familial adenomatous polyposis (FAP); somatic APC mutation is the earliest event in 80% of sporadic colorectal cancers

• VHL protein normally targets HIF-1α for ubiquitin-mediated degradation under normoxic conditions
• Loss of VHL → HIF-1α accumulates even under normoxia → constitutive activation of VEGF, PDGF, EPO, and other HIF target genes
• Causes clear cell renal cell carcinoma (germline: VHL disease; somatic: ~80% of sporadic ccRCC)

• Required for homologous recombination repair of double-strand DNA breaks
• Germline mutations: familial breast (~25% of familial cases), ovarian cancers
• BRCA-deficient cancers are particularly sensitive to PARP inhibitors (synthetic lethality - exploiting a DNA repair vulnerability)

• Phosphatase that dephosphorylates PIP3, opposing PI3K and negatively regulating AKT survival signaling
• Most commonly lost tumor suppressor after TP53; mutations in endometrial, prostate, glioblastoma, breast cancers
• Germline mutations: Cowden syndrome (hamartomas, breast/thyroid cancer risk)

• Encodes both p16 (inhibits CDK4/6) and p14ARF (inhibits MDM2, stabilizing p53)
• Deleted or silenced (promoter methylation) in a wide variety of cancers including melanoma, pancreatic cancer, NSCLC

• Mediators of TGF-β growth-inhibitory signaling
• Loss of SMAD4 in pancreatic and colorectal cancers deprives cells of TGF-β's growth-suppressive effects

5. Tumor Microenvironment (TME)

Key Cellular Components of the TME

• Most abundant stromal cells in many carcinomas (breast, pancreas, colon)
• Activated by TGF-β, PDGF from tumor cells
• Secrete ECM components (collagen, fibronectin), growth factors (HGF, IGF-1, FGF), and MMPs
• Create a desmoplastic stroma (e.g., in pancreatic cancer) that acts as a barrier to drug delivery
• Support tumor angiogenesis, invasion, and metastasis

• The most abundant immune cells in the TME of most solid tumors
• Polarized toward an M2-like (alternatively activated) phenotype by IL-4, IL-13, IL-10, and TGF-β from tumor cells
• M2 TAMs promote:
  • Tumor growth (secrete EGF, HGF, VEGF)
  • Immune evasion (produce IL-10, TGF-β; suppress CTL activity)
  • Angiogenesis (secrete VEGF, bFGF, CXCL8)
  • Matrix remodeling and invasion (secrete MMPs, cathepsins)
  • Phagocytosis of apoptotic tumor cells, clearing debris and limiting immunogenic cell death
• High TAM density correlates with poor prognosis in most cancers

• CD8+ CTLs: the principal anti-tumor immune effectors; can kill tumor cells presenting neoantigens on MHC class I. High TIL density correlates with better prognosis in melanoma, breast, colon (especially MSI-H) cancers.
• CD4+ helper T cells (Th1): produce IFN-γ, supporting CTL activity
• Regulatory T cells (Tregs): immunosuppressive; express FOXP3; suppress CTL and NK activity via IL-10, TGF-β, and CTLA-4/IL-2 consumption
• NK cells: innate immune effectors that kill tumor cells lacking MHC class I (KIR-mediated surveillance); suppressed by TGF-β and PD-L1 in the TME

• Immature myeloid cells (granulocytic and monocytic subtypes) that accumulate in blood and tumors
• Potently immunosuppressive via: arginase-1 (depletes arginine needed for T-cell activation), ROS production, TGF-β, IL-10
• Recruited by tumor-derived factors: VEGF, GM-CSF, M-CSF, CXCL5

• Tumor vessels are structurally abnormal: leaky, dilated, tortuous, poorly connected
• High interstitial pressure within the tumor impairs drug delivery
• Lymphangiogenesis (driven by VEGF-C, VEGF-D) facilitates lymphatic metastasis

• The ECM is extensively remodeled by cancer and stromal cells
• MMPs degrade basement membrane and ECM, releasing pro-angiogenic factors stored in the matrix (bFGF) and enabling tumor cell invasion
• Stiff, crosslinked collagen promotes mechanosignaling through integrins, activating oncogenic pathways (PI3K, FAK, YAP/TAZ)

Cancer-Enabling Inflammation

• Inflammatory cells produce reactive oxygen species (ROS) and reactive nitrogen species (RNS) that damage DNA
• Cytokines (TNF-α, IL-6) activate NF-κB in tumor cells, promoting survival, proliferation, and EMT
• Prostaglandins (via COX-2) promote tumor cell survival and angiogenesis
• Examples: H. pylori infection → gastric cancer; IBD → colorectal cancer; chronic hepatitis → HCC; asbestosis → mesothelioma

Immune Checkpoint Mechanisms in TME

• PD-1/PD-L1 axis: Tumor cells and MDSCs upregulate PD-L1, which engages PD-1 on CD8+ T cells, inducing T-cell exhaustion. This is the basis for checkpoint inhibitors (pembrolizumab, nivolumab).
• CTLA-4: Tregs and exhausted T cells express high levels of CTLA-4, competing with CD28 for CD80/CD86 costimulatory ligands. Ipilimumab targets CTLA-4.
• LAG-3, TIM-3, TIGIT: Additional co-inhibitory receptors that mediate T-cell exhaustion in the TME; emerging immunotherapy targets.

6. Laboratory Diagnosis of Cancer of Unknown Primary (CUP), Including IHC

Step 1: Morphological Assessment (H&E)

Step 2: Immunohistochemistry (IHC) - The Cornerstone of CUP Workup

• CK7+/CK20-: Lung, breast, ovary, endometrium, bladder, cholangiocarcinoma
• CK7-/CK20+: Colorectal carcinoma (classic pattern), Merkel cell carcinoma
• CK7+/CK20+: Urothelial, gastric, mucinous ovarian, cholangiocarcinoma
• CK7-/CK20-: Hepatocellular carcinoma, squamous cell carcinomas, renal cell carcinoma, prostate, adrenocortical

Step 3: Molecular and Ancillary Tests

• FISH for HER2 amplification in breast CUP with HER2 IHC 2+ (equivocal)
• EBER in-situ hybridization for EBV in undifferentiated nasopharyngeal carcinoma
• RET, ALK, ROS1 FISH in lung adenocarcinoma

• Multi-gene panels (e.g., Foundation One, MSK-IMPACT) can identify driver mutations that suggest tissue of origin:
  • KRAS G12C: lung adenocarcinoma
  • IDH1/2 R132: glioma or cholangiocarcinoma
  • BRAF V600E: melanoma, thyroid, colon
  • AR amplification: prostate
• Also identifies actionable alterations (MSI-H, TMB-H, NTRK fusions, BRCA mutations) enabling site-agnostic therapy (pembrolizumab for MSI-H, larotrectinib for NTRK)

• CancerSEEK, Tissue of Origin Test, CancerTYPE ID
• Analyze expression of hundreds of genes to compute probabilistic tissue-of-origin scores
• Sensitivity ~85% for correctly identifying primary site

• PSA (prostate), AFP (HCC/germ cell), β-hCG (germ cell/gestational trophoblastic), CA-125 (ovary), CA19-9 (pancreas/biliary), CEA (colorectal/lung), calcitonin (medullary thyroid)

• Identifies ultrastructural features: desmosomes (squamous), neurosecretory granules (neuroendocrine), melanosomes (melanoma), premelanosomes

Practical CUP Algorithm

Biopsy → H&E morphology
     ↓
Carcinoma? → Pan-CK, CD45, S100/HMB45, vimentin
     ↓
Adenocarcinoma → CK7/CK20 matrix → site-specific IHC panel
     ↓
Squamous → p40/p63 confirm → CK5/6 → site (lung, cervix, head/neck, skin)
     ↓
Neuroendocrine → Chromogranin, synaptophysin, Ki-67 (grade)
     ↓
Undifferentiated → SALL4/OCT4 (germ cell), CD45 (lymphoma), HMB45 (melanoma)
     ↓
Still uncertain → NGS/CGP + gene expression profiling

8. Molecular Techniques Used in the Pathology Laboratory

1. Fluorescence In Situ Hybridization (FISH)

• Principle: Fluorescently labeled DNA probes hybridize to complementary sequences in fixed tissue sections or cell preparations on glass slides
• Applications in pathology:
  • Gene amplification: HER2 amplification in breast cancer (determining HER2 status for trastuzumab eligibility)
  • Chromosomal translocations: BCR-ABL (CML), EML4-ALK (lung), SYT-SSX (synovial sarcoma), EWSR1 rearrangements (Ewing sarcoma, myxoid liposarcoma)
  • Chromosomal deletions: 13q (RB deletion), 17p (TP53 deletion in CLL), 1p/19q codeletion (oligodendroglioma)
  • Chromosomal gains/aneusomy: chromosome 3 in uveal melanoma
• Break-apart probes (detect gene rearrangement) vs. dual-fusion probes (detect specific fusion) vs. enumeration probes (copy number)

2. Polymerase Chain Reaction (PCR) and Variants

• Conventional PCR: amplifies specific DNA sequences; used to detect gene mutations, microbial DNA, viral integration (HPV, EBV, HBV)
• RT-PCR (Reverse Transcriptase PCR): detects gene expression by amplifying mRNA (converted to cDNA); used to detect fusion transcripts (BCR-ABL, ETV6-RUNX1 in ALL)
• Real-Time Quantitative PCR (qPCR): quantifies gene expression levels or copy numbers; used for minimal residual disease (MRD) monitoring in leukemia (BCR-ABL1 quantitation), HER2 copy number, CMV viral load
• Allele-Specific PCR / ARMS (Amplification Refractory Mutation System): detects specific point mutations (KRAS, BRAF, EGFR); high sensitivity for detecting mutations in heterogeneous tumor samples
• Digital PCR: counts individual DNA molecules; highest sensitivity for low-frequency variants; used for liquid biopsy (circulating tumor DNA)

3. Next-Generation Sequencing (NGS)

• Principle: massively parallel sequencing of millions of DNA fragments simultaneously; generates gigabytes of sequence data per run
• Types:
  • Targeted gene panels (e.g., 50-500 gene panels): sequencing hotspots and clinically relevant genes; most common in diagnostic labs
  • Whole-exome sequencing (WES): all ~22,000 protein-coding genes
  • Whole-genome sequencing (WGS): complete genome including non-coding regions; identifies structural variants, copy number alterations, mutational signatures
  • RNA sequencing (RNA-seq): transcriptome-wide gene expression; identifies fusion transcripts (NTRK, ROS1, RET, etc.)
• Applications:
  • Identifying actionable mutations: EGFR exon 19/21 (osimertinib), KRAS G12C (sotorasib), BRAF V600E (dabrafenib+trametinib), ALK/ROS1/RET fusions (targeted inhibitors)
  • MSI and TMB (tumor mutational burden) testing for immunotherapy eligibility
  • Tumor clonal evolution and resistance mechanisms
  • Hereditary cancer gene panels (BRCA1/2, Lynch syndrome genes, PALB2, CDH1, etc.)
  • Liquid biopsy: ctDNA from plasma samples

4. Chromogenic In Situ Hybridization (CISH) and Silver ISH (SISH)

• Similar to FISH but uses chromogenic (peroxidase/DAB) or silver deposition instead of fluorescence
• Can be performed on standard bright-field microscopy; signals are permanent (unlike FISH)
• Used for HER2 gene amplification (as alternative to FISH), HPV genotyping, EBV detection

5. Comparative Genomic Hybridization (CGH) and Array CGH

• Conventional CGH: compares copy number of entire tumor genome vs. normal genome
• Array CGH (aCGH): interrogates copy number at thousands of loci across the genome using microarrays
• Identifies chromosomal gains (amplifications) and losses (deletions) throughout the genome
• Used for pediatric tumors, constitutional chromosomal disorders, and comprehensive cancer profiling

6. DNA Methylation Analysis

• Bisulfite sequencing converts unmethylated cytosines to uracil; methylated cytosines resistant
• MGMT promoter methylation: silences the DNA repair gene in glioblastoma; predicts response to temozolomide chemotherapy
• MLH1 promoter methylation: indicates epigenetic silencing rather than mutation as cause of MSI
• DNA methylation profiling (Heidelberg classifier): classifies CNS tumors by methylation signature; now WHO-endorsed for CNS tumor classification

7. Gene Expression Profiling (Microarrays and RNA-seq)

• Measures expression levels of thousands of genes simultaneously
• Oncotype DX (21-gene recurrence score): predicts recurrence risk and chemotherapy benefit in ER+ HER2- early breast cancer
• PAM50/Prosigna: classifies breast cancer into intrinsic subtypes (Luminal A, Luminal B, HER2-enriched, Basal-like)
• Decipher (22-gene genomic classifier): prostate cancer recurrence risk after prostatectomy
• 70-gene signature (MammaPrint): breast cancer distant metastasis risk

8. Immunohistochemistry (IHC) - as a Molecular Surrogate

• Though tissue-based, IHC provides molecular information:
  • MMR protein IHC (MLH1, MSH2, MSH6, PMS2): screening for Lynch syndrome and MSI; loss of any protein expression triggers MSI PCR or NGS testing
  • p53 IHC: diffuse strong ("aberrant") staining = gain-of-function TP53 mutation; complete absence = nonsense/frameshift TP53 mutation; wild-type = scattered weak/moderate staining
  • PD-L1 IHC: companion diagnostic for immunotherapy eligibility (pembrolizumab in NSCLC, cervical, TNBC, etc.)
  • ALK IHC: highly sensitive/specific for ALK rearrangement in NSCLC; confirms before targeted therapy
  • BRAF V600E IHC (VE1 antibody): mutation-specific antibody for colorectal, thyroid, glioma screening

9. Flow Cytometry

• Measures multiple surface and intracellular markers simultaneously on single cells in suspension
• Applications:
  • Immunophenotyping of leukemias and lymphomas: defining lineage (B, T, NK, myeloid), maturation stage, aberrant antigen expression
  • Detecting clonal B-cell populations (light chain restriction - κ or λ excess)
  • MRD (minimal residual disease) monitoring after leukemia treatment - sensitivity 10⁻⁴ to 10⁻⁵
  • DNA ploidy and cell cycle analysis (S-phase fraction)

10. Cytogenetics (Karyotyping)

• G-banding of metaphase chromosomes from cultured tumor cells
• Identifies numerical abnormalities (monosomy, trisomy), structural rearrangements (translocations, inversions, deletions, amplifications as double minutes/HSRs)
• Essential in hematologic malignancies:
  • t(9;22) in CML, t(8;14) in Burkitt, t(15;17) in APL, t(14;18) in follicular lymphoma
  • Complex karyotype (≥3 abnormalities) in AML/MDS indicates poor prognosis
• Spectral karyotyping (SKY): uses multicolor FISH to visualize all 23 chromosome pairs simultaneously

11. Liquid Biopsy

• Analysis of cell-free DNA (cfDNA), circulating tumor DNA (ctDNA), circulating tumor cells (CTCs), exosomes from blood samples
• Applications: therapy monitoring, early relapse detection, resistance mutation identification (e.g., EGFR T790M in NSCLC after osimertinib), minimal residual disease, cancer screening
• Platforms: digital droplet PCR (ddPCR), ultra-deep NGS

Summary Table: Robbins Chapter 7 Key Concepts

Reference: Robbins & Cotran Pathologic Basis of Disease, 10th Edition (Kumar, Abbas, Aster). Chapter 7: Neoplasia, pp. 250-330. ISBN: 9780443264528.