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Pathology: Introduction & Neoplasia

Table of Contents

Interface of Pathology and Clinical Medicine .............................................................................................................1

Cellular Pathology I .....................................................................................................................................................2

Cellular Pathology II ....................................................................................................................................................4

Inflammation I – Acute Inflammation ........................................................................................................................6

Inflammation II – Chronic Inflammation ....................................................................................................................9

Biology of Human Neoplasia: Introduction and Overview ...................................................................................... 12

Pathology of Human Neoplasia (The Practical Issues) ............................................................................................ 15

Molecular genetics of cancer .................................................................................................................................. 18

Interface of Pathology and Clinical Medicine Pathology: pathos = suffering; logos = the study of Identical clinical presentations can be caused by dramatically different pathologies; different pathologies will require different treatments. Famous people with pathology include Hubert Humphrey, Sergi Grinchov, the anonymous roofer, Elvis, and JFK. Osler is responsible for 95% of all medical aphorisms.

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Cellular Pathology I Four pillars of pathology:

Etiology: what initiates a process?

Pathogenesis: what is its mechanism?

Morphology: how is it recognized?

Functional consequences: how does it produce disease? Disturbance of homeostasis:

Stress or increased demand can be met by adaptation; injurious stimuli may lead to cell injury / death.

Failure to adapt can lead to injury/death as well: e.g. adaptations to long-standing hypertension can predispose to sudden MI.

Hypoxia: reduction or absence of a normal oxygen supply to an organ (may result from ischemia = ischemic hypoxia)

Ischemia: reduction / absence of blood supply to an organ or tissue

Infarction: death of portion of tissue as result of ischemia (infarction = process, infarct = result)

White infarct: organs where there’s one blood supply (liver, kidneys, spleen) – wedge shaped infart downstream of blockage

Red infarct: main blood supply cut off, reperfused by secondary blood supply (e.g. lung) Mechanisms of cell injury (inter-dependent & synergistic):

Decrease in ATP o Example toxin: cyanide

Increased (or dec.) intracellular Ca+2 o Increase because Ca/Mg pumps shut down (↓ATP) o Leads to overactive enzymes (phospholipase, endonuclease, ATPase, protease) – damage

membrane, DNA, etc. o Example toxin: glutamate excitotoxicity in neurons

Reactive oxygen species – unpaired e- in outer orbit; leads to oxidative damage o Superoxide, H2O2, OH- or reactive nitrogen species too o Endogenous sources (metabolism, enzymes, ox-phos, inflammatory cells) o Exogenous sources (O2 toxicity, chemicals, radiation, reperfusion injury) o Usually in balance, but oxidative stress may occur if endogenous anti-oxidants overwhelmed

Aging, diabetes, alzheimer’s, smoking, cancer, atherosclerosis, etc. o Example toxin: acetaminophen in liver

Membrane damage if irreversible damange, considered the “point of no return” o Example toxin: complement from immune system

Reversible injury: shut down ox-phos, ↓ATP. Morphological features:

Swelling of organelles – ER, mito – and membrane blebbing (Na/K pumps shut down). Organelle changes a.k.a. hydropic/vacuolar degeneration

Clumping of chromatin (↑anaerobic glycolisis (lactic acid),↓pH, chromatin begins to clump)

Lipid deposition (↓ protein synthesis; lipids can’t be attached to proteins & build up in cell). A.k.a. fatty change (steatosis), seen in liver & myocardium

Irreversible when membrane damage starts to occur; time to “point of no return” depends on tissue.

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Reperfusion injury: previously ischemic area reperfused; inflammatory cells all enter at once; big influx of ROS

and calcium (pumps damaged) – may cause irreversible changes in cells. Necrosis: morphological changes in nucleus & cytoplasm occurring after cell death in a living tissue. (two key

points: cell now dead but host was alive when it happened). Features:

Eosinophilia (loss of RNA/ribosomes; proteins denatured). Looks more pink.

Nuclear features: pyknosis (dark, shrunken), karyorrhexis (broken down), karyolysis (totally dissolved)

Interstitial features: inflammation (need to be alive for this to happen Subtypes of necrosis:

Coagulative: after infarction (ischemic cell death) in solid tissues except brain. Most common. o Tissue architecture looks same, “tombstones” of hyper-eosinophilic cells (more pink) o Usually resolves as scar after neutrophils, macrophages scavange

Liquefactive: after infarction in brain. o Tissue architecture lost, complete hydrolysis / digestion of dead cells o Resolves by cyst/cavity formation o Abscess: Liquefactive necrosis as result of localized bacterial infections (fungal, parasitic)

Accumulation of neutrophils within abscess cavity (making hydrolytic enzymes) o Pus: dead neutrophils / cell debris

Requires surgical drainage

Caseous: “cheese-like”; after TB/fungal infections in immunocompetent individual

Granuloma: Necrotic center surrounded by rim of inflammatory cells

Fat necrosis: post-release of pancreatic lipase o Membrane lipids broken down to FFAs, add calcium = saponification (calcium/fat deposits)

Amount of tissue damaged permanently can depend on quickness of reperfusion (e.g. post-MI or stroke).

Functional consequences can vary for same etiology, pathology, etc.

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Cellular Pathology II

Hyperplasia: increase in the number of cells in tissue/organ. May or may not include hypertrophy.

Physiologic: e.g. compensatory hyperplasia (e.g. liver), lactating breast.

Pathologic o endometrial hyperplasia (pituitary-gonadal axis abnormalities, menorrhagia = heavy bleeding), o benign prostatic hyperplasia (includes secondary hypertrophy of bladder muscle)

Hypertrophy: increase in individual cell mass, leading to increase in organ mass. Reversible, response to stimulus

Physiologic: muscle hypertrophy after working out

Pathologic: hypertrophic myocardium (↑cytoplasm, ↑nucleus size = “boxcar nucleus”). Could be from chronic hypertension, aortic valve disease, or some other chronic hemodynamic overload.

Etiology: o hormone-induced (uterus & breast in pregnancy), o increased workload (pumping iron or pathologic / cardiac muscle) o genetic causes (myostatin mutation)

Atrophy: cellular shrinkage due to loss of substance.

Denervation (e.g. poliovirus infecting neurons innervating skeletal mm)

Disuse (e.g. hand with a cast on)

Hormonal: menopause (↓estrogen, endometrium from proliferative to cystic atrophy, can lead to irritation & atrophic vaginitis)

Senile atrophy (brain decreases in size with age)

Nutritional atrophy o Marasmus: protein-calorie malnutrition, swollen stomach b/c of lowered oncotic pressure o Cachexia: severe muscle wasting (AIDS, cancer, other chronic inflammation)

Cellular atrophy may include progressive cell loss so tissue / organ can shrink as well (gross scale atrophy too) Metaplasia: reversible replacement of one differentiated cell type by another differentiated cell type. Adaptive substitution (new cells can better withstand environment)

Smoking-associated squamous metaplasia – better able to withstand tobacco insult o Reserve cell metaplasia (change in reserve cell population, which are reprogrammed over time

to develop into squamous cells rather than columnar epithelium) o May undergo neoplastic progression (normal metaplasia dysplasia cancer) especially if

insult continues. o Example: Barrett esophagus (squamous columnar to withstand acid at gastroesophageal

junction). Dysplasia: epithelium starts to exhibit abnormal changes; pre-cancerous but mutations starting to occur Intracellular accumulations: cells can accumulate exogenous or endogenous substances

Anthracosis: universal finding in people who have lived in city. Black streaks = macrophages that phagocytosed carbon. No clinical importance

Lipofuscin: golden brown “wear-and-tear” pigment; tombstone of lipid peroxidation

Fatty change: absolute increase in intracellular lipids. Potentially reversible. Most common in liver o Causes: Morbid obesity (diffuse), alcohol abuse (may have central lobule sparing)

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o Does have clinical implications – can be irreversible if hepatocytes die fibrosis cirrhosis Apoptosis: programmed cell death.

Physiologic: embryogenesis, hormone-dependent (menstruation), mature tissue homeostasis

Pathologic: o Response to DNA damage from radiation, free radicals, etc. (via p53) o Viral infections (viral hepatitis) o Cytotoxic T-cell mediated injury (transplant rejection or autoimmune conditions)

Mediators (KNOW THIS) o Caspases: cysteine proteases that play essential role in execution phase of apoptosis. Require

activation from inactive form via activation cascade o Bcl-2: anti-apoptotic protein (but bcl-2 family contains both pro- and anti-apoptotic proteins) o p53: stops cell division in response to DNA damage to facilitate recovery; if recovery fails

apoptosis. Morphology of apoptosis:

Specific cells affected (necrosis = sheet of cells)

Organized process; systematic breakdown of DNA (necrosis = smear)

Inhibit apoptosis = facilitate tumorigenesis

HPV: carcinogen (squamous cell carcinoma of cervix) – HPV abrogates function of p53, p21

Follicular lymphoma – constitutive activation of Bcl-2

Characteristic Apoptosis Necrosis

Stimulus Usually physiologic Pathologic

Involvement Single cells Groups of cells

Chromatin Uniformly dense masses No pattern

DNA fragmentation Inter-nucleosomal Random

Cell morphology Apoptotic bodies Swelling, degen.

Inflammation Absent Present

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Inflammation I – Acute Inflammation Inflammation: a complex response of vascularized tissues to various stimuli, leading to the accumulation of

fluids and leukocytes in the extravascular tissues. Triggers include trauma, ischemia, neoplasm, infection, foreign matter, immune rxns, etc. Edema: excess of fluid in interstitial spaces or serous cavities (e.g. pleuroa, pericardium, peritoneum)

Transudate: edema with low protein content due to ↑ hydrostatic pressure

Exudate: edema with high protein content, often containing blood cells, due to ↑hydrostatic pressure and ↑ vascular permeability

o Serous: exudate with few inflammatory cells (pale yellow) o Serosanginous: exudate with erythrocytes (red tinged) o Fibrinous: contains large amounts of fibrin (after coagulation of clotting factors) o Purulent: high inflammatory cell content (often with bacterial infections) o Supperative: purulent exudate with significant pus (liquefactive necrosis)

What is inflammation trying to do? Deliver effector cells and molecules, provide physical barrier via microvascular coagulation to prevent spread, promote repair of offending tissue. Acute inflammation: early & immediate response (minutes to days).

Characteristics: o Edema (exudate of fluids & plasma proteins) o Emigration of leukocytes (esp. neutrophils)

Triple response of Lewis (histidine mediated): 1. Transient vasoconstriction 2. Wheal (fluid leakage) 3. Flare (vasodilation)

Steps: 1. Changes in vascular caliber and flow

Continuity equation: velocity = flow rate / cross sec. area (wider = slower velocity) Poiseuille’s law: flow rate increases with r4 (wider = more flow through) Bernoulli’s principle: velocity & pressure related inversely In acute inflammation: transient vasoconstriction of arterioles followed by vasodilation

of arterioles & capillary beds

Blood flow increases (Poiseuille’s): heat & redness

Blood velocity decreases (continuity): blood stasis

Hydrostatic pressure increases (Bernouilli): extravasation & exudate 2. Increased vascular permeability (leakage)

Plasma proteins lost so intravascular oncotic pressure drops (higher now in interstitum). Flow of water out (oncotic & hydrostatic pressure too) when normal balance disrupted

Results in tumor = swelling (edema) Mechanisms

Contraction of endothelial cells in venules (most common: separation of junctions; chemically mediated by histidine, reversible & short-lived – 15-30m)

Reorganization of cytoskeleton in endothelial cells, transcytosis, direct endothelial injury can play a role too

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3. Leukocytes extravasate & phagocytose Want to kill microbes, ingest offending agents, degrade necrotic tissue Lots of blood cells extravasate, not just leukocytes (RBC, platelets, etc) Glucocorticoids help reduce inflammation by decreasing extravasation

Role of leukocytes (extravasation & phagocytosis)

Granulocytes are key in acute inflammation

Neutrophils especially (eosinophils for allergies / parasitic infections, basophils & mast cells release histadine for allergic hypersensitivity, monocytes in chronic inflammation)

Time course: 1. Neutrophils (6-24h) 2. Monocytes / macrophages (24-48 hrs) 3. Lymphocytes (end, except viral infections where they can be first)

Sequence: 1. Extravasation

a. Margination: larger cells pushed to edge of vessel (RBC in central column) – pushed out even farther during inflammation

b. Rolling: tumbling & transient halting. i. Selectins: bone-marrow-derived & endothelial cell expression only

1. Slow down leukocyte under flow & signalling properties (rolling) 2. Ca dependent, carbohydrate binding proteins 3. Example: l-selectin on leukocyte, binds to GlyCam1 addressin (lymph node HEV)

ii. Addressins: expressed on endothelial cells on different sites, bind to homing receptors on lymphocytes

c. Adhesion: firm attachement to endothelial surfaces. Mediated by complementary molecules. i. Integrins: α/ß subunit heterodimers from IgG family with lots of types.

1. Classical examples are VLA4 (leukocyte) – VCAM1 (endothelial cell) and LFA1 (leukocyte) – ICAM1 (endothelial cell).

2. LFA1-ICAM1 binding is Mg dependent (↑affinity) and Ca dependent (↑avidity) d. Diapedesis: passage across endothelium through intercellular junctions

i. Happens in venules (no smooth mm in wall) ii. Endothelium to basement membrane, where they secrete collagenase to break down

e. Chemotaxis: direction to site of injury under influence of chemotactic agents; move using pseudopod

i. Exogenous: bacterial products ii. Endogenous: complement (c5a), lipoxygenase pathway products, chemokines (specific

for cell types, lots known) 2. Phagocytosis

a. What to eat? i. Opsonic phagocytosis: target covered by something (Fc receptors for Ab Fc segment,

complement receptors for C3b) ii. Non-opsonic phagocytosis: pathogen associated molecular patterns (PAMPs) (Mannose,

formyl-peptide, toll-like receptors for LPS, etc) b. Target identified, internalized via Ca-dependent process, phagosome fuses with lysosomes &

secretory vessels to destroy & excrete waste. 3. Microbial killing

a. Neutrophils make microbicidal free radicals i. NADPH oxidase reduces O2 to superoxide anion

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ii. Superoxide converted hydrogen peroxide by spontaneous dismutation iii. H2O2 is killing molecule (and other ROS/RNS)

b. Microbial response: catalase degrades H2O2 to H2O and O2 i. Pts with Chronic Granulomatous Disease lack NADPH oxidase system genes, susceptible

to infections by catalase positive microganisms Chemical mediators of inflammation: vasoactive amines (histamine, serotonin), plasma proteases (complement, kinins, clotting), arachadonic acid metabolites (prostaglandins, leukotrienes), cytokines – most involved in vascular permeability regulation too (no surprise) Complement puts holes in the target, kinin is involved in vasodilation and smooth mm relaxation, clotting involves fibrin depositing, cyclooxygenase is involved in prostaglandin formation (COX), cytokines & others get in play too.

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Inflammation II – Chronic Inflammation Outcomes of acute inflammation:

1. Complete resolution 2. Healing by connective tissue replacment (granulation tissue / organization; fibrosis) 3. Abcess formation 4. Chronic inflammation

Granulation tissue / organization

After big tissue damage / fibrin exudation

Buds of endothelial cells grow and canalize / anastamose

Macrophages migrate in

Myofibroblasts & fibroblasts appear, proliferate, form collagen fibers o Fibrosis: excessive deposition of collagen fibers

Appearance: packed with cells, juicy, capillaries, collagen with fibroblasts, macrophages removing stuff

Organization: granulation tissue replacing damaged tissue o Inflammation o Wound healing o Infarct o Thrombus

Fibroblasts: cytokine-mediated; produce collagen and ECM proteins Keloid: excessive formation of collagenous tissue resulting in raised area of scar tissue (broad bands of

collagen replacing normal dermal structures Cirrhosis in liver is result of inflammation fibrous tissue (collagen) process

Example of organization: pleura following pneumonia, fibrin exudate, forms pleural adhesion

Abcess: focal, localized collection of pus in a newly formed cavity

Pus in other cavities has different names (empyema in lungs, pyosalpinx in fallopian tubes, etc.)

Pus = purulent exudate with neutrophils, necrotic cells, edema fluid

Typically caused by Staphylococci (pyogenic bacteria)

Appearance: o Central: necrotic white cells & tissue cells o Around that: preserved neutrophils o Outer region: vascular dilation and fibroblastic proliferation (“pyogenic membrane”)

Can become walled off by connective tissue (body can’t access)

Can empty through fistula: pathologic channel connecting abcess to internal cavity / body surface

Phlegmon = cellulitis = opposite of an abcess (acute, overwhelming infection spreading along skin – S. aureus & group A streptococci)

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Chronic Inflammation:

a prolonged process where acute inflammation and destruction proceed at the same time as healing / immune response (balance)

Causes: anything that causes acute inflammation (if it persists), infections, autoimmunity (most common in US), alloimmunity (transplants), foreign materials (insoluble, inanimate) Clinical classification:

Primary: de novo cause (no clinically evident acute inflammation)

Secondary to acute inflammation Histological classification:

Macrophagic (diffuse or granulomatous), e.g. TB

Lymphocytic (diffuse or focal / follicle formation), e.g. autoimmunity

Supperative (lots of neutrophils, abcess formation) e.g. osteomyelitis Macrophages: most important cell in chronic inflammation

From bone marrow precursor

Circulate in blood as monocytes (half life of 1 day)

Migrate into tissue, transform into macrophages (half life of several months)

Roles of macrophages / monocytes: phagocytosis, induce immune reactions via antigen presentation, release signalling molecules

Activation: macrophages with increased inflammatory capacities; main function is phagocytosis but also release lots more substances (NO, ROS, proteases, cytokines, enzymes, grotwh factors, complement…)\

Can also cause significant tissue damage (hallmark of chronic inflammation) Macrophagic infiltration:

1. Granulomatous: macrophages arranged into compact masses (follicles); epitheloid appearance like a fence or barracade.

a. Granuloma = focal area of granulomatous inflammation. i. Small cluster of epitheloid cells surrounded by lymphocytes

ii. Caseation in middle, then epitheloid layer & macrophagic giant cells; ring of lymphocytes then fibrous tissue walling off on outside.

iii. E.g. tuberculosis b. Epitheloid cells: pale, pink, granular cytoplasm & indistinct cell boundaries; hypodense

elongated nucleous c. Giant cells: fusion of 6-8 macrophages (epitheloid); can contain 20+ small nuclei

i. Langhans giant cell (peripheral/horse-shoe nuclei arrangement): chronic immune granulomata like TB or sarcoidosis

ii. Foreign body giant cell (scattered nuclei throughout cytoplasm) – e.g. asbestosis d. Foreign body granuloma: particulate mater in middle (too large for phagocytosis by one Mφ) e. Immune granuloma: inducing cell-mediated immmunity, Mφ present to T-cells, T-cells produce

cytokines to transform Mφ to epitheloid & giant cells i. E.g. TB: granuloma (“tubercle”) caused by M. tuberculosis (acid-fast), usually caseating

f. GRANULOMA ≠ GRANULATION TISSUE 2. Non-granulomatous: diffuse spread of macrophages

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Lymphocytic infiltration: hallmark of autoimmune diseases

Collection of lymphocytes in an organ that doesn’t usually have them

Diffuse or focal lymphocytic infiltrations o Focal infiltrations: “ectopic follicles” – look just like lymph node follicles but elsewhere in body

B-cells in center, T-cells in cortex

Hashimoto’s thyroiditis: autoimmune reaction against thyroid (focal)

MS: collection of lymphocytes like follicle in brain Systemic effects of inflammation

Fever o Improve efficiency of leukocyte killing, impair replication of microorganisms o Coordinated by hypothalamus

Leukocytosis o WBC >11,000/uL blood o Accelerated release of cells from bone marrow (immature neutrophils = bands; “left shift”)

Bacterial infection: neutrophilia Viral infection: lymphocytosis Parasitic infection: eosinophilia

Leukopenia o WBC < 4,000 / uL o Typhoid fever, other infections, or when pts overwhelmed (disseminated TB, cancer, HIV)

INFLAMMATION SUMMARY

ACUTE CHRONIC DURATION Short (days) Long (months-years) ONSET Acute Insidious INFLAMMATORY CELLS Neutrophils, macrophages Macrophages, Lymphocytes, Fibroblasts VASCULAR CHANGES Vasodilation, leakage Angiogenesis (granulation tissue) EDEMA Yes Usually no CARDINAL CLINICAL SIGNS Yes Usually no TISSUE NECROSIS No Yes (ongoing) FIBROSIS No Yes (ongoing) SYSTEMIC EFFECTS High fever Low-grade fever, weight loss, anemia BLOOD CHANGES Neutrophilia, lymphocytosis Variable. Polyclonal

hypergammaglobulinemia

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Biology of Human Neoplasia: Introduction and Overview Neoplasia: clonal proliferation of cells with somatic genetic alterations and aberrant regulation of growth

Benign: don’t threaten life of neoplasm

Malignant (cancer): ability to invade into normal tissues and metastasize into distant tissues Neoplasms generally form masses (tumors) but some (e.g. pre-invasive or in situ neoplasms) don’t form visible masses. Key genetic defects in cancer cells:

activate genes that stimulate cell replication (e.g. growth factor receptor kinases)

inactivate genes that suppress cell replication Many cancers do have increased growth (↑mitotic figures & growth fraction = proportion of cycling cells). Others replicate at normal rate & suppress apoptosis (p53, bcl-2, BAX). So if tx only focuses on proliferating cells, may miss these that are suppressing apoptosis. Some of these pathways do both (regulate replication & apoptosis) – so these oncogenes can be very important; blocking their functions can even lead to regression of cancer via ↑apoptosis (“oncogene addiction”) Invasion & Metastasis Invasion:

1. Penetrate basement membrane, degrade ECM, migrate in stroma a. Cancer cells have active role (matrix metalloproteinases & other proteolytic enzymes) b. Invasion leads to tissue remodeling: stromal reaction (akin to chronic inflammation) c. Reactive stroma key to diagnosing invasion (is this tissue somewhere where it shouldn’t be?) d. Invasive cancer actively migrates (reprogramming of integrin gene expression changes in cell-

substrate adhesions & cytoskeletal dynamics) e. Microenvironmental cues (oxygen tension, pH) may guide cancer cells to specific stromal structures

2. Adapt to foreign environment a. Loss of cell-surface receptors (e.g. ↓e-cadherin, ↑other cadherins) b. Both begin to be able to bind to ECM & lose requirement to be bound to each other c. Called “epithelial-mesenchymal transition” (EMT) although not as complete as in embryology

(invasive cells still look like epithelial cells of origin)

Metastasis: spread of cancer to distant sites of body (not surgically treatable) 1. Vascular dissemination of malignant cells

a. Spread via lymphatics or blood vessels b. Pre-requisites: invasion into vascular space & ability to survive in circulation

i. Most non-hematopoietic cells / non-metastatic cancer cells can’t survive shear stress in circulation. But small % of cancer cells have “stem-like” properties & can survive in circulation.

ii. Most non-malignant cells undergo apoptosis when not attached to solid matrix (“anoikis”). But resistance to apoptosis already present in cancer cells.

c. Adhere to endothelium & extravasate through vascular walls (processes not well understood d. Usually inefficient (e.g. peritoneal-venous shunts in cancer pts don’t lead to widespread metastasis)

2. Growth of tumor in secondary site

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a. Determined in part by routes of vascular & lymphatic drainage (GI to mesenteric LN / liver, others to regional lymph nodes & lungs)

b. Not entirely dictated by drainage (breast, prostate, lung bone; breast/lung CNS) 3. Paget (1889) – “dependence of seed on the soil” (cancer cell on organ)

a. current research: chemokines from cancers & chemokine receptors in receptor organ tissue Cancer progression (classical paradigm): confined neoplasms invasive metastatic.

Classical: metastasis happens late

But there is big variability among neoplasms. Probably more likely that certain neoplasms are programmed to be aggressive or benign from the start depending on mutations. Even small tumors in some cancers can invade / metastasize early.

One explanation of why screening hasn’t resulted in huge reduction in cancer mortality Ability to modify the host environment

Tumor not just cancer cells: stroma, inflammatory cells, blood vessels

Angiogenesis: many cancers produce angiogenic substances (e.g. vascular endothelial growth factor, VEGF; fibroblastic growth factor, FGF); some also produce anti-angiogenic factors.

o Anti-VEGF Ab (bevacizumab) – Tx for some types of cancers o Low vascularity (hypoxic environment) helps some cancers (e.g. pancreatic) grow. Also makes

resistant to chemotherapeutic drugs because they don’t reach hypoxic areas

Suppression of immune surveillance: from “self” but altered enough to produce immune response o Cancer cells evade by secreting things (proteins to inhibit immune cells, cytokines /

prostaglandins to suppress immune response, decoy antigens) or express dummy receptors

Systemic effects of human cancers: May also secrete humoral factors that affect host physiology o Ectopic hormones (ACTH, parathyroid-related proteins) o Cachexins (e.g. TGF) – most not identified yet

Extended doubling potential of tumor cells

Normal cells: replicate, then eventually reach senescence (telomere shortening). Telomere important in chromosomal integrity

Cancer cells: immortality. Increased expression of telomerase to extend the telomeres. o Kicks in late so cancer cells still have reduced telomere length. o Could maybe detect cancer via telomerase expression or inhibit telomerase for therapy.

Genomic instability

Many somatic genetic mutations Multiple phenotypic alterations

Most cancer cells aneuploid (abnormal # & structure of chromosomes)

Continuous rearrangement as cancer cells divide o Shortening of telomeres – “anaphase bridging” where ends stick together in anaphase o Inadequate mitotic spindle checkpoint (imperfect alignment & segregation) o Problems maintaining structure: from defective DNA repair mechanisms (p53, BRCA1&2)

Leads to non-homologous recombination of broken chromosomes & translocatiosn o Defective mismatch repair: ↑mutations at sequence level

Increased microsatellite instability (MSI) Genomic instability important in carcinogenesis (need many mutations to make cancer) & development of

resistance to chemotherapy.

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Morphology: abnormal, with hyperchromatism (increased chromosomal material) & abnormal, irregular shape. Structurally abnormal mitosis.

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Pathology of Human Neoplasia (The Practical Issues) Classification & nomenclature

Prefixes = tissue of origin Benign tumors

-oma (chondroma = cartilage, adenoma = glandular epithelial)

Hepatoma, melanoma, astrocytoma are exceptions (malignant) Malignant tumors

-carcinoma = epithelial origin

-sarcoma = mesenchymal (stromal) origin Ways to characterize: 1. Patterns of differentiation (Epithelial, Mesenchymal, Hematopoetic, Melanocytic, Glial) 2. Sub-types: e.g. for epithelial neoplasm: squamous, glandular (adeno), basal/basaloid, transitional

(urothelial), undifferentiated. Each pattern of differentiation has its own sub-types 3. Morphology: papillary, cystic, polypoid, mucinous, etc. 4. Benign (have very minimal risk of progressing to malignancy) and malignant tumors

Borderline or low malignant potential tumors: don’t fall into these categories well

E.g. carcinoid tumor – neuroendocrine differentiation; respiratory / digestive systems; big range of malignancy.

Multiple patterns of differentiation:

epithelial + mesenchymal = fibroadenoma (benign) or carcinosarcoma (malignant).

Tetroma: more than one germ cell layer from pleuripotential cells Pattern of differentiation Benign Malignant

Epithelial

Glandular/ ductal epithelium Adenoma Adenocarcinoma

Squamous epithelium Squamous papilloma Squamous cell carcinoma or epidermoid carcinoma

Liver Hepatic adenoma Hepatoma (a.k.a., hepatocellular carcinoma)

Mesenchymal

Smooth muscle Leiomyoma Leiomyosarcoma

Adipocytes Lipoma Liposarcoma

Cartilage Chondroma Chondrosarcoma

Bone Osteoma Osteosarcoma

Endothelial hemangioma Hemangiosarcoma

Melanocytic Melanocytic nevus Melanoma

Glial Astrocytoma, ependymoma, oligodendroglioma

Hematopoetic Leukemia, lymphoma

Morphological characteristics of neoplasms:

Solid tissues form tumors (except in situ neoplasia, which is more spread out)

See below for benign vs. malignant cells

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These are descriptive characteristics, not rules (consideration of all features in context is important) Cytopathology: characterizing malignancy, etc. based on these features (e.g. Pap smear for aspirated cervical tissue) Clinical considerations for diagnosis of malignancy (based on clinical experience)

1. Site: smooth mm. tumor in uterus with certain # mitosis = leiomyoma, in colon = leiomyosarcoma 2. Gender: teratoma in ovary with benign appearance = benign course; in testis of adult male = high metastasis potential 3. Age: Benign-appearing teratoma in testis of child = benign course, benign appearance in adult male = malignant

Pre-invasive neoplasia (defies traditional definitions)

Tubular adenoma – precursor to colorectal cancer, low potential to invade (if excised, good prognosis)

Carcinoma in situ = “severe dysplasia of squamous mucosa” (e.g. cervix) – high % develops to invasive Grading & staging neoplasia

Grade: degree to which cells have malignant features. o Low grade = close to normal, high grade = large, irregular nuclei & atypical mitosis o Poorly differentiated / well differentiated (how well does it resemble normal tissue)? Well

differentiated = low grade, poorly differentiated = high grade o Standardized criteria (differentiation + nuclear features; nuclear dominate). Has variable

predictive validity depending on type of cancer

Stage: extent of spread of cancer. Better clinical predictor. o American Joint Committee on Cancer: TNM staging (Tumor, Lymph Nodes, Metastasis) o Combine information to make TNM grouping (T1, N2, M0 for example) – cut-offs depend on the

type of cancer o Grouped staging: 0, I-IV (0 = in situ, no invasion; IV = metastatic) – for surgery, etc.

Ancillary techniques Immunohistochemistry

No single marker but some are useful (e.g. p63 for normal basal cell layer in prostate; if missing = cancerous; AMACR overexpressed in most prostate cancers)

Mostly not helpful for benign vs malignant but can be used to phenotype tumor (e.g. heomatopoetic neoplasms – use on suspended cells post-flow-cytometry.

Detection of Cancer via Molecular Markers

Ideal situation: detect & monitor cancer via small markers with minimal invasiveness

Current: secreted proteins (tumor-specific antigens, e.g. prostate specific antigen/PSA)

Characteristics of benign cells Characteristics of malignant cells

Relatively low nuclear: cytoplasmic ratio Increased nuclear size (high N:C ratio)

Round nucleus, even distribution of chromatin, small or inconspicuous nucleoli

Irregular nuclear shape, irregular distribution of chromatin, prominent nucleoli

Maintenance of cellular polarity and differentiation Loss of cellular polarity and variable loss of differentiation

Mitoses are uncommon, are located in usual location (e.g., basal layer), and have typical appearance

Mitoses are common, located above basal cell layer, and have atypical appearance.

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o PSA: mortality has declined post-PSA introduction o Associated with over-Dx and over-Tx of disease o Some mixed studies on benefits in terms of mortality

Research: better early detection, monitoring disease (mass spec, DNA from cancer cells).

Still need tissue diagnosis before Tx currently (limits of sensitivity & specificity) Predictive markers / molecular classification

Want markers to predict response to therapy or subclassify tumors

E.g.: Estrogen receptor (ER) in breast cancer: predicts good response to anti-estrogen therapy & better prognosis.

Subclassify according to molecular features: e.g. measure multiple genes in parallel (microarray) to form prognostic indices & determine need for chemotherapy.

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Molecular genetics of cancer Theory of genetic basis: need social controls on cells; have a high mutational load of a complex organism. Average human gene mutated 1010 times in lifetime (almost all somatic) We handle our mutational load well:

Protect the germline cells (separation from somatic cells early in embryological development)

Innate resistance to tumorigenesis (single mutation inadequate)

Neoplasia requires accumulation of somatic mutations: a microevolutionary process

5-7 rate-limiting events needed (but how?)

Clonal selection theory: tumorigenesis occurs as serial expansion of successive clones of cells, punctuated by acquisition of certain mutations which give a cell and progeny a selective growth advantage over neighboring cells

o Why are so many mutations needed? Downregulation mechanisms protect cells (need multiple mutations to inactivate downregulatory syndromes & accumulate small effects to cause selective advantage)

o Clonal changes (present in all cells of a neoplasm) indicate important events o Genetic / epigenetic heterogeneity arises (even though genetic instability not universal in

neoplasms) – genetic instability just accelerates o Clone is population that derives from single cell; offspring (subclones) compete to see who can

dominate neoplasm (with selection). Otherwise you’d just end up with heterogenous group & benign neoplasm. Have to select each time one by one or else tumor mass would be huge

o Subsets with worse prognosis = those with more mutations o Neoplasms arise from chance events so genetic profile varies from pt to pt (individualize

therapy) Pediatric tumors may be exception (arise in window of opportunity & don’t resemble

adults: maybe need fewer mutations & not as many steps)

Mitogenesis is as important as mutagenesis in tumorigenesis o Not just environmental exposure to mutagens, but also inflammation & regeneratory processes

Changes in production of stem cells & ability to differentiate are key for neoplasia (commonly mutated)

Frequently mutated genes: 1. Dominant oncogenes: function activated by mutations

o Think about the signal transduction system from outside to nucleus o Growth factors o Growth factor receptors (e.g. EGFR) o Signal transducers (e.g. RAS, ABL-BCR) o Nuclear oncoproteins o Agonists of apoptosis (BCL-2) o Antagonists of tumor-suppressors (e.g. antagonists of p53)

2. Tumor-suppressor genes: function inactivated by mutations (selective advantage) o Cell-cell, cell-ECM, differentiation-inducing interactions (e.g. E-cadherin) o Cytoskeletal o Regulators of signal transduction o Cell-division cycle regulation (e.g. p53) o Apoptosis (ultimate negative regulation: p53, BAX)

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o Chromatin structure 3. DNA-maintenance genes: genes inactivated by mutations, often before tumorigenesis (need second hit

for selective advantage) o DNA repair genes (e.g. xeroderma pigmentosum genes) o Chromosome stability genes (BRCA2, etc.)

4. Passenger mutations: have no special meaning in the neoplasm

Types of mutations:

Amplification: overproduction of proteins

Rearrangement / translocation: fusion of two genes from different proteins or oncogene placed behind strong promoter

Small mutation: e.g. point mutation, can activate or inactivate gene

Large deletion: often a second hit – cause inactivation of suppressor gene, or loss of heterozygosity (LOH), exposing first hit’s mutation

Viral insertion: can allow viral oncogenes to continue to be expressed

Telomere shortening: can cause genetic instability, deletions, translocations. Re-activation in malignancy helps prevent extreme of this process (cell death) in malignant tumor cells

Inherited syndromes: can be due to germline mutations in: 1. Dominant oncogenes (examples are rare: embryonic lethal?) 2. Tumor suppressor genes (more common). Two-hit model: maybe the first hit is inherited, increasing

rate of neoplasm’s occurrence. E.g. familial adenomatous polyposis (FAP) a. For instance: recessive gene but get LOH with second hit b. Other examples: hereditary retinoblastoma (RB1), familial breast/ovarian cancer (BRCA 1), Li-

Fraumeni syndrome (p53 – lots of cancers possible) 3. DNA maintenance genes

a. True examples of higher mutation rates (“genomic instability”) b. Hard to find “genetic instability” in lab, but some conditions do have true chromosomal

instability c. Examples:

i. Xeroderma pigmentosa (inadequate repair of UV-induced DNA damage if have 2 mutant copies of the gene)

ii. Hereditary nonpolyposis colorectal carcinoma (can be heterozygous) – inherited cause of cancer susceptibility

iii. Ataxia telangiectasia (2 mutant copies) iv. Fanconi anemia & familial breast/ovarian/pancreatic cancer (BRCA2). Two mutant

copies = highest risk, Fanconi anemia. One mutant copy still increases risk (get LOH in neoplasm)

4. Susceptibility genes: may be very common & are being studied currently

Rational therapy: Old model: screen all kinds of toxins for ability to kill cancer cells in culture New model: look for specific biochemical properties New ideas: augment deficient function(p53 – hard); replace function (hard without gene therapy working); inactivate a function (Gleevec – very successful); take advantage of neoplastic defect; re-express genes; augment immune responses

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Acquired drug resistance via mutations: from mutations in drug-binding pocket; mutations causing compensatory increase in activity, or mutations eliminating cell’s toxic response

Carcinogens

Dietary / environmental: o can use Ames assay (expose to potential mutagen; count colonies on plate that have mutated,

subtract background rate), others (cheaper in bacteria, really expensive in animal models). o End up only screening things that are pretty certain to be carcinogenic o Some are suspected carcinogens o Example: Aflatoxin causes p53 mutations in hepatocellular carcinomas in Africa & China

Infectious causes: o Indirect mechanism: mitogenesis & inflammation (e.g. HBV & hepatocellular carcinoma, H.

pylori & gastric cancer) o Direct mechanism: viral proteins that inactivate tumor-suppressor genes (e.g. HPV & cervical

cancer) Non-mutated genes can also play a role (may be over- or under-expressed in neoplasms & provide good background for neoplastic development

What is a neoplasm “A clone of cells distinguished from other tissues by autonomous growth and somatic mutations”

Mutations in growth-controlling genes

Supporting, reactive tissues accompany tumor growth

Grow in conditions that would otherwise be limiting Caveats:

All neoplasms have been found to have somatic mutations

Inciting stimulus usually not shown for neoplasms

Neoplasms often do control their own proliferation, but control is altered & cell # increases (evidence: most neoplasms are benign)

Other masses & proliferations o Keloids, developmental abnormalities, granulation tissue, synovitis, etc. o As long as it’s not clonal, it’s not a neoplasm

Neoplasms are not always masses (e.g. leukemias, etc.)

Neoplasms are not just a proliferative abnormality (this would just be hyperplasia) but rather a large increase in stem cell # (clonal)

Mutations in growth controlling genes can be inherited rather than acquired (insufficient to cause neoplasms on their own). ADDITIONAL SOMATIC MUTATIONS ALWAYS REQUIRED.

FAP VS HNPCC (Familial Adenomatous Polyposis vs. hereditary nonpolyposis colorectal cancer)

FAP: first change occurs quickly (lots of early adenomas) but it takes around 20 years to accumulate more hits

HNPCC: first change occurs slowly, but fast progression afterwards (2 years) – harder to treat