Molecular Biology

Hallmarks of cancer (Hanahan-Weinberg)

• Sustained proliferative signaling: RTK/RAS/RAF/MEK/ERK pathway hyperactivation.
• Evading growth suppressors: Rb, p53 inactivation.
• Resisting apoptosis (resisting cell death): Bcl-2 family, MDM2.
• Replicative immortality: telomerase (hTERT) reactivation.
• Angiogenesis: VEGF/HIF.
• Invasion & metastasis: EMT, matrix metalloproteinases.
• Immune evasion (avoiding immune destruction): PD-L1, MHC down-regulation.
• Enabling characteristic: genome instability: MMR, HRD, BRCA, TP53 loss.
• Metabolic reprogramming (deregulating cellular energetics): Warburg effect (aerobic glycolysis).
• Enabling characteristic: tumor-promoting inflammation.

Framework (multistep tumorigenesis): Hanahan and Weinberg first proposed 6 core hallmarks (2000); a 2011 update added 2 emerging hallmarks now considered core (deregulating cellular energetics, avoiding immune destruction) plus 2 enabling characteristics (genomic instability, tumor-promoting inflammation). A 2022 revision (Hanahan) added further emerging hallmarks and enabling characteristics. Capabilities can be acquired in any order, and one mutation may confer more than one.

Foundations: genes, expression, and epigenetics

• Genome: ~3 billion nucleotides across 23 chromosomes; only ~1% is protein-coding. ~20,400 protein-coding genes, but the proteome spans 250,000 to 1 million proteoforms (from alternative splicing and posttranslational modification).
• Central dogma: transcription (DNA → premRNA, introns spliced out) → translation (ribosomes read codons) → posttranslational modification (phosphorylation, acetylation, ubiquitination, methylation) controls protein activity, stability, and localization.
• Regulatory elements: promoters (site of initiation) and enhancers (increase transcription); recognized by transcription factors, coactivators, and corepressors.
• Noncoding RNAs:
  • miRNAs (~25 nt): decrease expression of target mRNAs; >2,500 identified; aberrant expression linked to neoplasia.
  • lncRNAs (>200 nt): diverse roles (silencing transcription, chromatin modulation, splicing). Examples: NKILA, MALAT1, TERC (telomere length), XIST (X inactivation).
• Epigenetics (heritable change without altering DNA sequence), two main mechanisms:
  • DNA methylation: DNA methyltransferases add methyl groups to CG dinucleotides; promoter methylation represses transcription. Silences tumor suppressors (e.g., MLH1, CDKN2A/p16). Targeted by hypomethylating agents (azacitidine, decitabine) in MDS/AML.
  • Histone modification: DNA wraps histone octamers (nucleosomes); acetylation (active transcription) vs deacetylation (repression); methylation site-dependent. Targeted by HDAC inhibitors (vorinostat, panobinostat, belinostat) and EZH2 inhibitor tazemetostat (follicular lymphoma).

Oncogenes vs tumor suppressors

• Oncogenes (gain-of-function, dominant): MYC, RAS, RAF, ALK, EGFR, HER2. A single mutated allele is sufficient (proto-oncogene → oncogene).
• Tumor suppressors (loss-of-function, recessive, two-hit Knudson): RB1, TP53, APC, BRCA1/2, PTEN, NF1. Typically one allele mutated and the other lost (loss of heterozygosity, LOH).
• Mechanisms of oncogene activation: point mutation (KRAS, EGFR, neomorphic IDH1/2), amplification (HER2, MYCN, AR in castration-resistant prostate; can be intrachromosomal or extrachromosomal "double minutes"), and translocation/fusion.
• Exceptions to Knudson (biallelic loss of function not required): haploinsufficiency (one allele loss suffices) and dominant-negative mutations (mutant inhibits wild-type protein, e.g., FBXW7, SPOP, TP53); note promoter methylation (e.g., CDKN2A) can serve as one of the two hits rather than a true exception.
• Opposed oncogene/tumor suppressor pair: PIK3CA (kinase that adds phosphate to phosphatidylinositol) vs PTEN (phosphatase that removes it).

Key signaling pathways

• RTK / MAPK (RAS/RAF/MEK/ERK): growth factor binds receptor tyrosine kinase → dimerization, autophosphorylation → downstream signaling. RAS cycles between active GTP-bound and inactive GDP-bound states; GEFs activate, GAPs (e.g., NF1) inactivate. Oncogenic RAS mutations (codons 12, 13) impair GTP hydrolysis → constitutive signaling. Three RAS genes: KRAS, NRAS, HRAS. BRAF V600E in ~50% of melanoma.
  • BRAF: vemurafenib, dabrafenib, encorafenib.
  • MEK: trametinib, cobimetinib, binimetinib.
  • KRAS G12C: sotorasib, adagrasib.
• PI3K / AKT / mTOR: PI3K activation (one of the most common cancer mutations) drives AKT, affecting growth, survival, protein synthesis, and metabolism. PTEN (tumor suppressor) opposes PI3K; loss increases AKT activity.
  • PI3Kα: alpelisib, inavolisib.
  • AKT: capivasertib.
  • mTOR: everolimus, temsirolimus.
• Cell cycle (CDK4/6 - cyclin D - Rb): CDKs (catalytic) + cyclins (regulatory) drive phase transitions; CDK inhibitors (INK4, p27Kip1) restrain them. CCND1 amplified/translocated (MCL, myeloma); CCNE1 amplified (ovarian). pRB is the prototype Knudson tumor suppressor (retinoblastoma), also lost in SCLC and bladder cancer. Palbociclib, ribociclib, abemaciclib with antiestrogens for HR+ breast cancer.
• Wnt / β-catenin: APC loss stabilizes β-catenin → nuclear TCF-driven transcription. APC mutations in familial adenomatous polyposis and most sporadic CRC (early event); CTNNB1 in desmoid.
• Hedgehog: ligand → PTCH1 → SMO → GLI transcription factors. PTCH1/SMO in BCC, medulloblastoma. Vismodegib, sonidegib, glasdegib; arsenic trioxide inhibits GLI.
• Notch: ligand-induced cleavage releases intracellular domain that activates transcription; MYC is a key mediator. Activating NOTCH1 in up to 50% of T-ALL (γ-secretase inhibitors); nirogacestat in desmoid. Can act as tumor suppressor in squamous carcinomas.
• JAK/STAT: MPNs (ruxolitinib, fedratinib, momelotinib).
• Differentiation block (APL): t(15;17) PML-RARA fusion acts as dominant-negative on RARα targets and blocks differentiation; ATRA reverses the block; core-binding factor translocations similarly impair hematopoietic differentiation.

Cell-cycle control & checkpoints

• Phases: G1 (growth, integrates mitogenic signals, restriction point), S (DNA synthesis), G2 (growth), M (mitosis). Mutations affecting S-phase commitment are among the most common in cancer.
• Cell-cycle checkpoints (distinct from immune checkpoints): monitor DNA replication/mitosis and halt the cycle for repair or trigger apoptosis/senescence. Sensors/mediators → transducers (ATM, ATR, then CHK1, CHK2) → effectors (p53, Cdc25).
• Mitotic spindle assembly checkpoint: ensures equal chromosome segregation via kinetochore-spindle attachment (MAD2/Cdc20/APC-C). A single unattached kinetochore holds the "wait" signal. Chromosomal instability (CIN) is common but rarely from kinetochore gene mutations; targeted indirectly by taxanes and vinca alkaloids.

Apoptosis

• Intrinsic (mitochondrial) pathway: triggered by radiotherapy, chemotherapy, cellular stress; mitochondrial membrane changes release cytochrome C → caspase cascade → DNA fragmentation.
• Extrinsic (death receptor) pathway: death ligands (TNFα, FAS ligand) bind death receptors (TNFR1, FAS) → caspase activation.
• BCL-2 family: pro-apoptotic vs anti-apoptotic regulators. BCL2 first identified from t(14;18) in follicular lymphoma. BCL-2 overexpression prevents MYC-driven apoptosis (aggressive "double-hit" lymphomas). Venetoclax (BH3 mimetic) approved in CLL and AML.
• TP53 loss is the most common mutation impairing apoptosis; PI3K-AKT also suppresses apoptosis.
• Other cell-death programs: autophagy, necrosis, entosis, and ferroptosis (iron-dependent, discovered 2012).

p53 pathway

• TP53 ("guardian of the genome") is the most frequently mutated tumor suppressor; germline loss causes Li-Fraumeni syndrome (early sarcomas, breast, brain, adrenocortical carcinoma, leukemia).
• p53 is a transcription factor activated by DNA damage/replication stress → cell-cycle arrest, apoptosis, or senescence. Usually inactivated by loss of one copy plus mutation of the other.
• MDM2: E3 ubiquitin ligase induced by p53 that degrades p53 (negative feedback); overexpressed/amplified in many cancers. ARF tumor suppressor binds MDM2 to protect p53; ARF is induced by MYC and is frequently deleted.
• Specific TP53 mutations can confer gain-of-function; destabilizing mutants such as Y220C can be refolded by investigational reactivators.

DNA repair pathways & DNA damage response (DDR)

• Homologous recombination (HR): high-fidelity double-strand break (DSB) repair. BRCA1/2, PALB2, ATM, RAD51 and paralogs are HR genes, but PARPi sensitivity is not uniform: BRCA1/2 and PALB2 have strong evidence, whereas ATM alterations alone do not confer BRCA-type sensitivity (TBCRC 048, TRITON3 ATM subgroup).
• Non-homologous end joining (NHEJ): error-prone DSB repair.
• Mismatch repair (MMR): corrects single-base mismatches/small indels. MLH1, MSH2, MSH6, PMS2 (Lynch). MSH2-MSH6 (MutSalpha) recognizes the mismatch; MLH1-PMS2 (MutLalpha) is recruited and PMS2 nicks the daughter strand (strand discrimination via PCNA and replication nicks). Defects → microsatellite instability (MSI-H = dMMR), hypermutation, and immune-checkpoint responsiveness. Sporadic dMMR is usually MLH1 promoter hypermethylation.
• Nucleotide excision repair (NER): repairs UV dimers/bulky adducts (global and transcription-coupled). Germline defects: xeroderma pigmentosum (~10,000-fold nonmelanoma skin cancer and ~2,000-fold melanoma risk under age 20), Cockayne syndrome, trichothiodystrophy.
• Base excision repair (BER): excises damaged bases; Biallelic MUTYH germline mutations (autosomal recessive, MUTYH-associated polyposis) predispose to colorectal cancer; monoallelic carriers are not managed as MAP.
• Double-strand-break syndromes: ataxia telangiectasia (ATM, a PI3K-like kinase phosphorylating CHK2, TP53, BRCA1, NBS1); Fanconi anemia (FANCD2/FANCI monoubiquitination; FANCD1 = BRCA2; hypersensitivity to crosslinkers like mitomycin C); Nijmegen breakage syndrome (NBS1; MRE11-NBS1-RAD50 complex).
• Synthetic lethality: HRD (BRCA) + PARP inhibitor = death. Somatic and germline DDR/BRCA alterations confer comparable PARP-inhibitor sensitivity; predictive value depends on the gene and biallelic/functional loss, not inherited vs acquired origin. DDR defects also predict cisplatin sensitivity.

Telomeres & replicative immortality

• Telomeres: repetitive nucleotide sequences that cap and protect chromosome ends. They shorten with each division; when critically short, cells normally undergo replicative senescence; if senescence is bypassed, further shortening causes end-to-end fusions, genomic instability, and telomere crisis, during which most cells die.
• Cancer immortality: tumors maintain telomere length, usually by reactivating telomerase (hTERT), detectable in 85 to 90% of cancers; the remainder use recombination-based "alternative lengthening of telomeres" (ALT).

Angiogenesis

• Tumors outgrowing their blood supply recruit new vessels by upregulating pro-angiogenic factors (VEGF, FGF) and reducing inhibitory signals; hypoxia via HIF stabilization is a key driver.
• Hypoxia: HIF-1α/HIF-2α stabilization → angiogenesis, glycolysis; VHL loss (RCC, HIF-2α driven) is a classic upstream lesion. Targeted by belzutifan (HIF-2α inhibitor) for VHL.
• Therapeutics: anti-VEGF antibody (bevacizumab), VEGFR small-molecule inhibitors.

Ubiquitin-proteasome system

• Proteins tagged (via E1, E2, E3 enzymes) with polyubiquitin chains are degraded by the proteasome. E3 ligases are key oncogenes/tumor suppressors.
• FBXW7 (degrades cyclin E, c-MYC, Notch, c-JUN): commonly mutated tumor suppressor (T-ALL, endometrial). SPOP: mutated in prostate/endometrial cancer. VHL: loss causes VHL syndrome and clear cell RCC (VHL targets HIF-1α and HIF-2α for degradation; HIF-2α is the key ccRCC driver). MDM2: overexpressed E3 that degrades p53.
• Drugs: proteasome inhibitors (bortezomib, carfilzomib, ixazomib) in myeloma; thalidomide analogs redirect cereblon E3 ligase; PROTACs (proteolysis-targeting chimeras) selectively degrade target proteins.

Common cancer-driver mutations

• TP53: "guardian of the genome"; most commonly mutated gene in cancer.
• KRAS: pancreatic (90%), CRC, lung adeno (~25%).
• BRAF: melanoma (50%), thyroid PTC, CRC, hairy cell leukemia (V600E).
• PIK3CA: breast, endometrial.
• EGFR: lung adenocarcinoma (15% in US, ~50% Asian non-smokers).
• MYC: Burkitt (8;14), neuroblastoma (MYCN amplification).
• ALK: NSCLC, ALCL, IMT, neuroblastoma.
• BCR-ABL (t(9;22)): CML, B-ALL.
• PML-RARA (t(15;17)): APL.
• IDH1/IDH2: glioma, AML, cholangiocarcinoma, chondrosarcoma. Neomorphic enzyme produces the oncometabolite 2-hydroxyglutarate, causing DNA hypermethylation.

Translocations to know

• t(8;14) MYC-IGH: Burkitt.
• t(14;18) BCL2-IGH: follicular lymphoma.
• t(11;14) CCND1-IGH: mantle cell lymphoma.
• t(11;22) EWSR1-FLI1: Ewing sarcoma.
• t(X;18) SS18-SSX: synovial sarcoma.
• t(2;13) PAX3-FOXO1: alveolar RMS.
• t(12;21) ETV6-RUNX1: pediatric B-ALL.
• inv(16) CBFB-MYH11: AML M4eo.
• t(8;21) RUNX1-RUNX1T1: AML M2.
• t(15;17) PML-RARA: APL.
• TMPRSS2-ERG/ETV1: prostate cancer (androgen-driven ETS expression).

Tumor microenvironment & metabolism

• Hypoxia: HIF-1α and HIF-2α stabilization drive angiogenesis and glycolysis; HIF-2α is the key ccRCC driver, targeted by belzutifan (HIF-2α inhibitor).
• Warburg effect: aerobic glycolysis (lactate even with O2); basis of FDG-PET.
• Metabolic targets: IDH (ivosidenib, enasidenib), PHGDH, glutaminase.
• Cancer-associated fibroblasts (CAFs), tumor-associated macrophages (TAMs).
• PD-L1, IDO, MDSCs: immune evasion mechanisms.

Infectious agents as cancer drivers

• ~15% of cancers worldwide (>2 million people/year), mostly chronic viral infection.
• HPV: squamous carcinomas of head/neck, cervix, penis, vulva, vagina, anus (E6 degrades p53; E7 degrades RB).
• EBV: Burkitt and other lymphomas, nasopharyngeal carcinoma, some gastric cancers.
• HBV/HCV: hepatocellular carcinoma. HIV / HHV-8 (KSHV): Kaposi sarcoma. HTLV-1: adult T-cell lymphoma. Merkel cell polyomavirus: Merkel cell carcinoma.
• Non-viral: H. pylori (gastric cancer, gastric MALT lymphoma); liver flukes (hepatobiliary cancer). Prevention: HPV and HBV vaccination; H. pylori eradication.

Detection technologies

• PCR / RT-PCR / qPCR: amplification-based; qPCR monitors BCR-ABL transcript in CML and MRD. Digital droplet PCR detects rare mutations (e.g., ESR1, ALK, PIK3CA in circulating DNA).
• NGS: massively parallel short reads (~100 to 200 bp) mapped to reference; detects point mutations, indels, copy-number changes, fusions, TMB, MSI. Variant allele frequency reflects the mutant read fraction. Genome sequencing fell from ~$1 million (2008) to ~$1,000 (2019).
• Cytogenetics/FISH: karyotype and probe-based detection of amplifications (HER2), fusions (BCR-ABL, EML4-ALK), and residual disease.
• Protein methods: Western blot, ELISA, IHC (HER2, ER/PR, MMR proteins, p16 for HPV), flow cytometry (immunophenotyping, MRD), CyTOF (up to ~100 proteins by mass cytometry), MS-based proteomics/metabolomics.
• Single-cell & spatial genomics: barcoding/UMIs, microfluidics (10X, Drop-seq); tumor atlases (Human Tumor Atlas Network, HTAN); normal-tissue atlases (HuBMAP).
• Research tools: RNAi (siRNA) and CRISPR/Cas9 (2020 Nobel) for gene knockout, editing, and genome-wide screens (e.g., PTPN2 in immune evasion; saturation editing of BRCA1 variants).

Biomarker testing modalities

• IHC: protein expression (HER2, ER/PR, PD-L1, MMR proteins, BAP1, BRCA1/PALB2).
• FISH/ISH: gene amplification (HER2, MET), translocations (ALK, ROS1, BCR::ABL1).
• NGS (next-generation sequencing): comprehensive, point mutations, indels, fusions, CNV, TMB, MSI.
• Homologous recombination deficiency (HRD) testing: BRCA mutation + genomic scar score (loss of heterozygosity, telomeric allelic imbalance, large-scale state transitions). Genomic-scar HRD score is a validated PARPi predictor mainly in ovarian cancer (PAOLA-1). In breast, pancreatic, and prostate cancer, PARPi selection is based on BRCA1/2 (and, in prostate, selected HRR gene) alterations, not a generic HRD scar score. Myriad myChoice CDx, FoundationOne CDx (LOH score).
• Liquid biopsy (ctDNA):
  • Genotyping when tissue inadequate or unavailable.
  • MRD detection: post-curative-intent surgery (DYNAMIC for stage II CRC, BESPOKE, GALAXY).
  • Resistance monitoring: detect emergent mutations (e.g., ESR1, T790M, KRAS G12C secondary mutations).
  • Treatment response: ctDNA dynamics correlate with outcome.
• Multi-cancer early detection (MCED): Galleri (Grail) leads category, not yet FDA-approved (PMA under review; advisory committee favorable Sep 2026), commercially available LDT. PATHFINDER Lancet 2023.
• Companion diagnostics (CDx): FDA-approved tests required for specific drugs (Therascreen for KRAS G12C, FoundationOne CDx, Ventana for HER2/PD-L1).

2024-2026 targeted-therapy pathway advances

• Menin-KMT2A inhibitors: revumenib (Revuforj), FDA Nov 2024 for r/r KMT2A-r acute leukemia (AUGMENT-101: ORR 63%, CR/CRh 22%); revumenib expanded Oct 2025 to r/r NPM1-mut AML; ziftomenib (Komzifti), FDA Nov 2025 for r/r NPM1-mut AML.
• MTAP-null tumors + PRMT5-selective inhibitors: MTAP deletion co-occurs with CDKN2A loss in ~15% of solid tumors; MRTX1719 (Mirati) and AMG 193 phase 1/2, synthetic-lethal PRMT5-MTA inhibitors. MTAP IHC/NGS increasingly reported.
• KRAS pan-inhibitors: RMC-6236 (daraxonrasib), RAS(ON) multi-selective inhibitor, FDA Aug 2026 (Rasonque) for previously treated metastatic PDAC (RASolute 302); RMC-9805 (KRAS G12D-selective); divarasib (KRAS G12C, next-gen).
• CLDN18.2 targeting: zolbetuximab (Vyloy), FDA Oct 2024 for CLDN18.2+ HER2- gastric/GEJ (SPOTLIGHT, GLOW). IHC 2+/3+ 75%+ cells threshold. Emerging CAR-T (CT041) and ADCs (IBI343).
• Mutant TP53 reactivators: rezatapopt (PC14586) selective for TP53 Y220C, phase 2 PYNNACLE ongoing; first pan-tumor mutant-p53 targeted therapy.
• KAT6A/B inhibitor: PF-07248144 for HR+/HER2- MBC, phase 1 (Mayer SABCS 2024).
• CDK2/CDK4 selective inhibitors: INX-315 (selective CDK2 inhibitor; CCNE1-amplified, CDK4/6i-resistant tumors); PF-07220060 (CDK4-selective, spares CDK6, less cytopenia); CDK2i for CCNE1-amplified tumors.
• ctDNA MRD assays: Signatera, Guardant Reveal, NeXT Personal; strongly prognostic, but escalation based on ctDNA remains investigational: DYNAMIC supported de-escalation in stage II colon cancer, BESPOKE is observational, and ALTAIR missed its primary DFS endpoint.

High-yield molecular biology pearls

• Knudson "two-hit" for tumor suppressors (retinoblastoma/RB paradigm).
• TP53 is "guardian of the genome," most commonly mutated gene; MDM2 degrades it, ARF protects it.
• Synthetic lethality = HRD (BRCA) + PARP-i.
• MMR defect = MSI-H = dMMR = IO-responsive; sporadic dMMR is usually MLH1 methylation.
• IDH mutation = 2-HG accumulation = oncometabolite.
• Telomerase reactivation gives replicative immortality in 85 to 90% of cancers.
• Translocations (Burkitt 8;14, follicular 14;18, MCL 11;14, Ewing 11;22, APL 15;17).
• Belzutifan: HIF-2α inhibitor for VHL.
• Warburg effect = aerobic glycolysis, basis of FDG-PET.
• Cell-cycle checkpoints (ATM/ATR, CHK1/2, p53) are distinct from immune checkpoints.