Diagnostic Methods and Molecular Basis of Cancer

Examples of Chemical Carcinogens

ALKYLATING AGENTS - Cyclophosphamide, Bulsulfan AROMATIC HYDROCARBONS - Cigarette Smoke AZO DYES - β-Naphthylamine (Aniline Dye) - Bladder Cancer NATURALLY OCCURING CARCINOGENS - Aflatoxin B₁ produced by Aspergillus flavus - HCC in Africa NITROSAMINES AND AMIDES - Synthesized in GI tract from ingested Nitrites - Gastric Cancer ASBESTOS, VINYL CHLORIDE, NICKEL

Xeroderma Pigmentosum

Autosomal recessive mutation that results in defective Nucleotide Excision Repair that predisposes risk for cancers following exposure to UV light. They have abnormal pigmentation & neurological abnormalities.

Epstein-Barr Virus

Binds to CR CD21 and infects B-Cells, Immortalizing them and causing Lymphoma. Associated with African Burkitt Lymphoma, B Cell Lymphoma in immunosuppressed patients, some HL, Nasopharyngeal Carcinoma.

Ultraviolet Radiation Carcinogenesis

Causes Squamous Cell and Basal Cell Carcinomas Increased Risk depends on the Type and Intensity of Radiation as well as the Quantity of Melanin in the skin. UVB is the most potent mutagen and functions by forming Pyrimidine Dimers that are repaired by Nucleotide Excision Repair (if defective, Xeroderma Pigmentosum)

Carcinogenic Agents

Chemicals Radiant Energy Oncogenic Viruses and Others

Tumor Supressor Genes

Code for proteins that downregulate cell division Recessive, Loss of Function mutations

DNA Repair Flow Chart and Examples

DNA Repair Mutations allow for Mutations in other genes. EXAMPLES: Xeroderma Pigmentosum Ataxia Telangiectasia Hereditary Nonpolyposis Colon Cancer (HNPCC)

Microbial Carcinogenesis

DNA VIRUSES - HPV, EBV, HBV RNA VIRUSES - HTLV-1 BACTERIA - Helicobacter Pylori

EBV & Nasopharyngeal Carcinoma

Endemic in specific areas and 100% contain EBV DNA. However, restricted geographic distribution indicates other factors are involved.

Retinoblastoma and Development

FAMILIAL (40% of Cases) - One mutation is inherited, one is somatically acquired SPORADIC (60% of Cases) - Both mutations are acquired

Hereditary Nonpolyposis Colon Cancer (HNPCC) aka Lynch Syndrome

Familial carcinomas of the colon caused by defective DNA Mismatch Repair, which produces Microsatellite Instability.

Categories of Genes Responsible for Tumoigenesis

GAIN OF FUNCTION: Proto-Oncogenes - Growth Factors, Growth Factor Receptors, Transcription Factors, Signal Transducers LOSS OF FUNCTION: Tumor Suppressor Genes - Growth Inhibitor Factors and Growth Inhibitor Factor Receptors, Signal Transducer Inhibitors DISRUPTION OF NORMAL CELL DEATH: Apoptosis Genes - Located in Mitochondria DISRUPTION OF NORMAL DNA REPAIR: DNA Repair Genes - Located in Nucleus

Oncogenes

Genetic alterations (Gain of Function Mutations) to Proto-Oncogenes that produce abnormal protein products, Oncoproteins, that do not respond to their regulatory elements.

Helicobacter pylori & Gastric Lymphomas

H. pylori infection detected in most Gastric Lymphomas. Tumors arise in MALT (MALTomas). The B cells that give rise to these tumors reside in the Marginal Zone of lymphoid follicle (Marginal Zone Lymphoma). Infection initially generates H. pylori-reactive T cells, which activate a polyclonal population of B cells. Over time the B cells become monoclonal & independent. Treatment of infection w/abx usually causes lymphoma regression.

Chemical Carcinogenesis

INITIATION - Exposure of cells to sufficient dose of Carcinogenic Agent that causes Irreversible DNA Damage. The Agents act either DIRECTLY or INDIRECTLY (procarinogens), where they require conversion in vivo to produce ultimate carcinogens capable of transforming cells. PROMOTION - Agent that causes cell to proliferate so that previous DNA Damage (in initiated cells) becomes a permanent change (Promoters are nontumorigenic on their own). In this case, cellular changes do not affect DNA & are reversible.

Initiation and Promotion Pic

In order to get Tumor Development, you need *application of the Initiator followed by application of the Promoter*. In case 6, you see that you need continuous application of the Promoter to develop tumors.

RAS Oncogene

Inactive RAS is bound to GDP Growth Factor stimulation causes Ras activation by exchanging GDP for GTP, which activates the MAP-Kianse Pathway, leading to Cell Division. This pathway is normally active for a very brief period, as GTP is quickly hydrolyzed to GDP. This functions NORMALLY, but a mutation that prevents hydrolysis of GTP to GDP will cause continuous cell division. Mechanism: Mutant RAS binds GAP (GTPase-Activating Proteins), but GTPase activity fails.

Helicobacter pylori & Gastric Adenocarcinomas

Increased epithelial cell proliferation combined with chronic inflammation (ROS) leads to cancer development. Chronic gastritis → Gastric atrophy → Intestinal metaplasia → Dysplasia → Cancer Occurs over decades and only occurs in a small % of affected individuals.

EBV & B Cell Lymphoma

Mainly in AIDS patients & patients on long term immunosuppressive therapy. Tumors start out as polyclonal but may develop into monoclonal neoplasms. Tumors may regress w/relaxation of immunosuppression.

Retinoblastoma Susceptibility (RB) Protein

Molecular On-Off switch for the cell cycle: QUIESCENT CELLS: Hypophosphorylated Rb binds to E2F and DP1. This complex of E2F/DP1/RB then binds to promoters of E2F-Responsive Genes and silences them. DIVIDING CELLS: Growth factors ↑ Cyclins D & E → CyclinD-CDK4 and CyclinE-CDK2 hyperphosphorylate RB allowing for activation of E2F leading to transcription. This permits progression from G₁ → S Phase.

HBV

Most HCCs are associated w/HBV or HBC, but role is unclear. Highest rates of HCC where HBV & HBC are endemic. HVB & HBC genomes do NOT encode viral oncoproteins. HBV does integrate, but without a consistent pattern.

EBV & Burkitt Lymphoma

Most common childhood cancer in Central Africa & New Guinea >90% of African tumors carry EBV genome & all patients have Ab to viral capsid antigens. HOWEVER, other factors are involved. Not everyone w/EBV gets BL & it's not often found in BLs outside of Africa. All BL tumors have t(8,14)

Li-Fraumeni Syndrome

Mutated p53 leads to increased risk for many Cancers

HTLV-1

Only retrovirus that definitely causes cancer. Endemic in Japan & Caribbean basin. Transmitted by sex, blood products or breastfeeding and has tropism for CD4 T cells. Leukemia develops in 3-5% after latent period of many decades. It is also associated w/Tropical Spastic Paraparesis. Unclear mechanism, but virus gene tax does cause T cell proliferation.

MSI Testing

PCR Amplification of Microsatellite regions. Compare allelic profiles from matching normal & tumor tissues. Presence of alleles in tumor sample not found in normal sample in same individual indicates MSI.

Proto-Oncogenes

Promote normal growth and differentiation (code for proteins that trigger cell division) Tightly Regulated

bcl-2

Prototypical Gene that regulates Apoptosis; functions by sequestering Cytochrome C in the Mitochondria in order to PREVENT Apoptosis Normally, other members of the bcl-2 family like bax promote the release of Cytochrome C, creating a ratio of bcl-2:bax that will determine whether or not a cell responds to Apoptotic Stimuli. Overexpression of bcl-2 leads to cell survival (Common in B Cell Follicular Lymphomas)

Mechanisms of Proto-Oncogene Activation

QUANTITATIVE - Increase in amount of Oncogene product or production in inappropriate cell types (Insertion of Viral Genome, Chromosomal Translocation, Gene Amplification) QUALITATIVE - Alteration of Nucleotide sequence confers new properties (Point mutations, Chromosomal Translocation)

Cyclins, CDKs & CDK Inhibitors

Regulate progression through the various phases of the Cell Cycle

Chromosome 8, 14 Translocation

Seen in most Burkitt Lymphomas Results in fusion of Heavy Chain Ig (C Heavy) with Myc Oncogene.

Acquired Capabilities of Cells Leading to Cancer

Self-Sufficiency in Growth Signals Insensitivity to Anti-Growth Signals Evading Apoptosis Limitless Replicative Potential Sustained Angiogenesis Tissue Invasion and Metastasis

Microsatellites and Microsatellite Instability (MSI)

Short repeated DNA sequences composed of repeat units that are 1 to 6 base pairs in length. Microsatellites are distributed throughout the Human Genome. MSI is a change in length of Microsatellites resulting from Insertion/Deletion. Normally this would be corrected by DNA Mismatch Repair Enzymes.

G₀ Stage of Cell Cycle

Stage of Cell Cycle in which most non-dividing cells are located

Genetic Basis of Carcinogenesis

Stems from Non-Lethal Cell Damage that can be either acquired or inherited. Multiple *successive mutations* in genes controlling cell growth, differentiation, apoptosis or DNA repair lead to a single progenitor cell that becomes immortal.

Human Papilloma Virus

TYPES: 16 and 18 associated with Invasive Squamous Cell Carcinoma, Cervical Dysplasia & Carcinoma in-situ; 6 and 11 only associated with Warts IMPORTANT PROTEINS: E6 and E7 disable p53, p21 and RB

Telomeres and Telomerase

Telomeres are repeated sequences at the ends of the Chromosomes that get shortened with every cell division. Once Telomeres reach a certain length, they signal the cell to stop dividing and senesce. Telomerase is an enzyme that protects the ends of Chromosomes from shortening. GERM CELLS: Have Telomerase SOMATIC CELLS: No Telomerase In some cells, Telomerase can be reactivated. Maintenance of telomere length inhibits senescence, resulting in a cell with a growth advantage & increased risk of acquiring mutations.

Philidelphia Chromosome

Translocation between Chromosomes 9 and 22 that leads to a mutant Bcr/Abl protein that is the cause of Chronic Myelogenous Leukemia

p53

Tumor Suppressor Gene that is mutated in 50% of cancers.

APC Gene (Adenomatous Polyposis Coli)

Tumor Suppressor Gene that sequesters β-Catenin and prevents it from activating Myc Transcription. Patients heterozygous for mutant APC develop many polyps that eventually will progress to colon cancer.

BRCA-1 and BRCA-2

Tumor Suppressor Genes that function in DNA Repair; BRCA-1 → Breast and Ovarian Cancer BRCA-2 → Male Breast, Ovarian Cancer

Knudson's Two Hit Hypothesis

Two independent mutations need to affect both chromosomes, in order for the Loss of Tumor Suppressing Functions. Often, the First Hit is inherited and the Second Hit is acquired through Somatic Mutations.

Heterogeneity of Tumors

While once cell may seed a tumor, additional genetic alterations can create subclones that possess growth advantages (they are selected for). In this way, the tumor can have multiple different types of cells.

Ionizing Radiation Carcinogenesis

X-Rays, γ-Rays, α-Particles, β-Particles, Protons, Neutrons all cause cancer. Organ Susceptibility: HIGH - Myelopoietic Tissue, Thyroid MODERATE - Breast, Lungs, Salivary Gland LOW - Skin, Bone, GI Tract