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CHAPTER 7
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NEOPLASIA

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• Cardiovascular diseases are the leading cause of death in the US. Cancer is second
• What are the 2 basic components of benign and malignant tumors?
1. Parenchyma (clonal neoplastic cells)
2. Reactive stroma
!
Benign Tumors
• Adenoma: benign epithelial neoplasm derived from glands, though they may or may not form
glandular structures
• Papilloma: benign epithelial neoplasms producing microscopically visible finger-like or warty
projections from epithelial surfaces
• Polyp: a neoplasm that produces a macroscopically visible projection above a mucosal surface
• Hamartoma: disorganized, benign tissue that is normally found at the site of the nodule
• Choristoma: a heterotopic rest of cells (a well developed, normally organized structure found in
a location that is unexpected), normally a congenital anomaly
!
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CHARACTERISTICS OF BENIGN AND MALIGNANT NEOPLASMS
Differentiation: the extent to which neoplastic parenchymal cells resemble the corresponding
normal parenchymal cells, both morphologically and functionally.
!
Anaplasia: lack of differentiation
• Major features include:
1. Pleomorphism
2. Abnormal nuclear morphology (hyperchromatic, increased N:C ratio (normal 1:4),
irregular nuclear shape, clumped chromatin, large nucleoli)
3. Mitoses, especially atypical ones
4. Loss of polarity
• Other changes include formation of tumor giant cells, tumor necrosis
!
Metaplasia: replacement of one cell type with another type (nearly always associate with tissue
damage, repair or regeneration)
!
Dysplasia: disordered growth, characterized by a loss of uniformity of the individual cells as well as
a loss in their architectural orientation
!
• It is believed that most cancers arise form less mature cells with stem cell like properties and in
well differentiated tumors the cancer cells retain that capacity to differentiate while in poorly
differentiated tumors this capacity is lost.

Rates of Growth
• The smallest clinically detectable mass is 1 gram of tissue (109 cells).
• Only 10 additional doubling cycles are required to form a tumor weighing 1 kg (the maximal
size compatible with life).
• By the time a solid tumor is clinically detected it has completed most of its life cycle.
!
Q: What are the 3 main factors determining the rate of tumor growth?
1. The doubling time of the tumor cells
 2. The fraction of tumor cells in the replicative pool
3. The rate of cell death
!
Q: What is the growth fraction?
• The proportion of cells in the proliferative pool
• This has a profound effect on chemo
• Debulking tumor tends to shift more tumor cells into the cell cycle making them more
susceptible to chemotherapy
• Tumors with a small pool of dividing cells tend to be chemo resistant
!
Progressive tumor growth and rate of growth ultimately depend on:
• Excess of cell production over cell loss
!
In general the growth rate of tumors correlates with their level of differentiation
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Cancer Stem Cells and Cancer Cell Lineages
• Cancers are immortal with limitless proliferative capacity and hence must contain “stemlike”
properties
• If cancer stem cells are essential for tumor survival then these must be eliminated to cure the
patient
• It is hypothesized that cancer stem cells have a high intrinsic resistance to therapy
because of their low division rates and expression of proteins that resist chemo (MDR-1)
• Genes and pathways that maintain cancer stem cells are the same as those that regulate
normal stem cell homeostasis
!
Q: What are tumor initiating cells?
• Cells that allow a human tumor to grow and maintain itself indefinitely when transplanted
into an immunodeficient mouse
• They have been identified in several cancers including GBM, breast, and colon cancer
!
Metastasis
Q: What are the 2 most reliable features separating benign from malignant tumors?
1. Metastasis
2. Invasiveness
!
Q: Define metastasis: tumor implants discontinuous with the primary tumor.
30% of newly diagnosed patients with cancer present with mets.
!
Q: What are the 3 main pathways of cancer spread?
1. Direct seeding of body cavities/surfaces
2. Lymphatic spread – most common pathway for carcinomas
3. Hematogenous spread – most common pathway for sarcomas
!
• The pattern of lymphatic spread normally follows the natural routes of drainage, however, skip
mets may occur because of venous-lymphatic anastomosis or lymphatic channels have been
obliterated by inflammation of previous radiation.
!
Q: Define sentinel node: the first node in a regional lymphatic basin that receives lymph flow from
the primary tumor
!
• In hematogenous spread veins are more easily penetrated than arteries. Tumor cells often grow
in the first capillary bed they encounter (liver and lungs most frequent). Cancers arising close to
the paravertebral plexus will go to the spine (thyroid, prostate).
!
• The natural pathways of venous and lymphatic drainage do not entirely explain the distribution
of mets. Tumors also have tissue specific homing factor
EPIDEMIOLOGY
Genetic Predisposition to Cancer
What are the 3 general categories of genetic predisposition to cancer?
1. AD cancer syndromes
• Normally due to a point mutation of a tumor suppressor gene
• second allele is silenced in somatic cells due to deletion or recombination (eg.
Retinoblastoma, LiFraumeni, BRCA1/2, FAP)
• For each syndrome tumors tend to arise at specific sites and tissues
• Often associated with a specific marker phenotype
• Incomplete penetrance and variable expressivity can occur
• HNPCC is the most common cancer predisposition syndrome
2. AR cancer syndromes
• Generally due to defects in DNA repair with resulting DNA instability (eg. Bloom
syndrome, AT)
3. Familial cancers
• No clearly defined pattern of transmission
• Early age at onset, tumors arising in 2 or more close relatives, multiple/bilateral
tumors
• Multifactorial inheritance
• Not associated with a marker phenotype
!
• There is also the close interaction between genetic and non-genetic factors
• Genotype can significantly influence likelihood of developing environmentally induced cancers
• Inherited variations (polymorphisms) of enzymes that metabolize procarcinogens to carcinogens
(p450 enzymes) can influence susceptibility of cancer
!
Nonhereditary Predisposing Conditions
Chronic Inflammation and Cancer:
• Ulcerative colitis, HP gastritis, viral hepatitis, chronic pancreatitis
• Chronic inflammation may increase the pool of tissue stem cells and expose them to
mutagens
• Many mediators in inflammation will promote cell survival even in the face of DNA damage
• These mechanisms favor survival of the organism in the short term
• The expression of cox-2 is increased in colon cancer
!
Precancerous Conditions:
• Certain non-neoplastic disorders have a well defined association with cancer
• Eg: pernicious anemia, solar keratosis of skin chronic UC, leukoplakia of oral cavity, vulva
and penis
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MOLECULAR BASIS OF CANCER
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• Nonlethal genetic damage lies at heart of carcinogenesis
• may be caused by the environment, viral, or randomly during normal cell replication
• Tumors are formed from the clonal expansion of a single precursor cell that has incurred genetic
damage
!
Q: What are the 4 classes of regulatory genes that are the principle targets of DNA damage in
cancer?
1. Proto-oncogenes (dominant)
2. Tumor suppressor genes (recessive)
3. Genes that regulate apoptosis
4. Genes involved in DNA repair (mutator phenotype)
!
• Carcinogenesis: multistep process at phenotypic and genetic levels, resulting from accumulation
of multiple mutations
• Eve though most malignant tumors are monoclonal in origin, by the time they become clinically
evident their constituent cells are extremely heterogeneous
!!
Essential alterations for malignant transformation:
1. Self sufficiency in growth signals (can proliferate w/o external stimuli)
2. Insensitivity to growth inhibitory signals
3. Evasion of apoptosis
4. Limitless replicative potential
5. Sustained angiogenesis
6. Ability to invade and metastasize
7. Defects in DNA repair
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SELF-SUFFICIENCY IN GROWTH SIGNALS: ONCOGENES
Oncogenes: genes that promote autonomous cell growth in cancer cells, proto-oncogenes are the
unmutated counterparts
• Oncogenes are generally constitutively active.
!
Proteins encoded by proto-oncogenes can be growth factors, growth factor receptors, signal
transducers, transcription factors, or cell cycle regulators.
• Tumors tend to synthesize growth factors that they are responsive to (autocrine loop), not
necessarily due to a mutated growth factor (eg. Mutated RAS causes increased production of
growth factors)
• Ongogenic versions of growth factor receptors are active without growth factor binding
• Often increased receptor expression results from gene amplification
The RAS oncogene (signal transducing oncoprotein)
• Part of the G-protein family
• Point mutations in the RAS family of
genes is the most common abnormality
of proto-oncogenes in human tumors
• RAS activation and inactivation depends
on nucleotide exchange (GDP – GTP)
and acceleration of GTP hydrolysis by
GAPs (GTPase activating proteins)
• Point mutations in RAS typically involve
the GTP binding pocket or the
enzymatic region with intrinsic GTPase
activity
• Mutations of GAPs also lead to
prolonged RAS activation (eg.
Neurofibromin 1 is a GAP that is
mutated in NF1)
• Downstream members of the signaling
cascade may also be mutated
• Dysregulation of the MAPK pathway
alone is not sufficient for carcinogenesis
(eg. 80% of benign nevi contain mutations in BRAF)
!
!
The MYC Oncogene
• MYC is the most commonly involved transcription factor in human tumors
• The range of activities modulated by MYC is very broad
• Histone acetylation, reduced cell adhesion, increased cell motility, increased protein
synthesis, decreased proteinase activity, etc
• Genomic mapping of MYC binding sites has identified thousands of different sites
• There is little overlap of MYC target genes in different cancers
• MYC may allow a replicating cell to bypass checkpoints
• MYC can act to reprogram cells into pluripotent stem cells
• MYC is translocated in Burkitt lymphoma and upregulated in neuroblastoma (N-MYC), and SCC
of lung (L-MYC)

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Cyclins and Cyclin-Dependent Kinases
• Cyclins and CDKs are overexpressed in many
cancers
• CDK inhibitors exert negative control over the cell
cycle.
• CIP/WAF CDKIs include p21, p27, p57
• INK4 family of CDKIs include p15, p16,
p18, p19
• CDKIs are frequently mutated or silenced in
cancers
!
• Defects in cell cycle checkpoints are also major
causes of genetic instability of cancer cells.
• RB required for G1/S checkpoint
• P53 is required for the G1/S checkpoint and a
main component of the G2/M checkpoint.
!
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INSENSITIVITY TO GROWTH INHIBITION AND ESCAPE FROM SENESCENCE: TUMOR
SUPPRESSOR GENES
• Oncogenes drive proliferation
• Tumor suppressor genes stop proliferation
!
Q: Name 4 ways in which tumor suppressor genes inhibit proliferation:
1. Many tumor suppressors will inhibit proliferation in response to any source of genotoxic
stress (p53, RB)
2. Expression of an oncogene in an otherwise normal cell leads to senescence
3. Some tumor suppressors will ultimately activate apoptosis
4. Tumor suppressor genes can force cells to differentiate and lose replicative potential
!
Q: What is Knudson’s hypothesis?
• 2 hits involving both RB alleles (chromosome 13) are required to produce retinoblastoma
• In familial cases, 1 defective allele is inherited
• Because only a single mutation is required for loss of RB function, the familial variant is
considered autosomal dominant
• In sporadic cases, both alleles must be mutated
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RB Protein
• RB is active and hypophosphorylated in quiescent cells
• RB is inactive and hyperphosphorylated during progression of cell cycle (G1/S)
• Once cells pass the G1/S checkpoint they are obligated to complete mitosis
• In G1, cells can still exit the cell cycle
• RB is a key player in the G1/S checkpoint
• In the active hypophosphorylated state, RB binds to E2F (E2F is the transcription factor for cyclin
E, which is required for DNA replication, S phase)
• Active RB also recruits other proteins that alter the promoter regions of E2F dependent genes
(such as cyclin E) making transcription of these genes inefficient
• RB is inactivated by cyclin D – CDK4/6 complexes in response to mitogenic signals (eg.
Signaling from EGF)
• Activation can be blocked by various CDK inhibitors (eg. P16)
• Loss of RB or mutations in the E2F binding pocket lead to uncontrolled progression through the
cell cycle
• Mutations in p16, cyclin D, and CDK4 can mimic the results of RB mutations (causes
dysregulation of the RB pathway)
• Oncogenic viruses also act by dysregulating the RB pathway
• SV40, polyomavirus, adenovirus, and HPV E7 protein all bind to the hypophosphorylated
form of RB, displacing E2F from its binding pocket
Q: Name 4 ways that the RB protein can become inactivated in human cancers:
1. Mutations in the E2F binding pocket
2. Mutations of p16
3. Mutations of cyclin D
4. Mutations of CDK4
!
P53: Guardian of the Genome
• P53 is located on chromosome 17 and is the most common genetic alteration in human tumors
• P53 is a transcription factor that controls hundreds of genes
• 80% of p53 point mutations in human cancers are located in the DNA binding domain
• In tumors without a p53 mutation, p53 can be functionally inactivated through a mutation in
another gene that normally regulates p53 function (eg. MDM2 – normal stimulates degradation
of p53)
• P53 is normally associated with MDM2 in healthy cells, which gives p53 a short half life
• When a cell is stressed, p53 is released form MDM2 and has an increased half life
• Genes activated by p53 fall into 2 broad categories:
1. Genes that cause cell cycle arrest
2. Genes that cause apoptosis (BAX and PUMA)
• A third broad category is emerging:
3. MiRNAs that repress pro-proliferative and anti-apoptotic genes (mir34 MiRNAs
are required for p53 function)
• Targets for mir34 include cyclins and BCL2
• If the damaged DNA can be repaired during cell cycle arrest the cell returns to a normal
state, if not p53 induces apoptosis or senescence
• Oncogenic viruses will also interfere with p53 activity
• HPV E6 will bind to and promote degradation of p53
!
Q: What are 3 ways that p53 prevent neoplastic transformation of cells?
1. Activation of quiescence of temporary cell cycle arrest
2. Activation of senescence (permanent arrest)
3. Triggering apoptosis
 • The key initiators of the DNA damage pathway are ATM (ataxia-telangiectasia mutated) and
ATR
• ATM and ATR activate p53 and DNA-repair proteins
• The cell cycle is paused in late G1 (G1/S checkpoint) by p53 activation of p21 (which inhibits
the phosphorylation of RB and release of E2F) and DNA repair is attempted
• Senescence is a permanent cell cycle arrest that can be mediated by p53
• The mechanisms of senescence are unclear but likely involve irreversible changes in the
DNA preventing transcription of pro-proliferative genes such as E2F
• There is an element of type-specific response based on cell type (some cells enter
senescence while others readily undergo apoptosis)
• The decision to repair the DNA or enter apoptosis is partly determined by the level of p53
• The repair pathway is stimulated first
• If enough p53 accumulates, the apoptotic pathway is stimulated
• The mechanism is not entirely clear
• The control of apoptosis also has therapeutic implications
• Tumors that retain normal p53 are more likely to respond to RT and chemo since these
tumors will proceed to apoptosis following severe damage from these treatments
• Tumors with p53 mutations are relatively resistant to RT and chemo
• Other p53 family members include p63 and p73
• P53 is ubiquitously expressed while p63 and 73 are more tissue specific
• P63 is essential for the differentiation of stratified squam epithelium
• P73 has strong pro-apoptotic effects
• Different isoforms of these genes are also expressed
• In basal breast cancers, p53 is commonly mutated and a dominant negative isoform of
p63 is commonly expressed that antagonizes the pro-apoptotic activity of p73
!
APC/Beta Catenin Pathway
• APC is located on chromosome 5 and is a family of tumor suppressor genes that function to
down regulate mitogenic signals
• APC is part of the WNT signaling pathway – major roles in cell adhesion and polarity
• WNT signals through a family of receptors called frizzled
• In the absence of WNT signaling, APC causes degradation of B-catenin
• APC mops up free B-catenin in the cytoplasm and directs it for proteasomal destruction by
ubiquitination
• Signalling by WNT blocks APC allowing B-catenin to go to the nucleus where it increases
the transcription of pro-proliferation genes such as c-myc and cyclin D1
• If cells lose functional APC, they behave as if they are under continuous WNT signalling
• B-catenin normally binds to the cytoplasmic tail of E-cadherin (E-cad maintains cell-cell
contact)
• Loss of cell-cell contact disrupts the connection between E-cad and B-catenin allowing B-
catenin to go to the nucleus
• This would be the appropriate response to injury to try and repair the wound
• As the wound heals, E-cad establishes cell connections and sequesters B-Catenin
Name 2 ways that the WNT pathway is altered in malignant cells:
1. Mutations of APC result in increased levels of cytoplasmic B-catenin
2. Mutations in E-cadherin result in increased levels of B-catenin, mutations in cadherins also
result in discohesiveness favoring invasion and metastasis
!
INK1a/ARF (CDKN2A gene locus) encodes p16/CDK1 and p14/ARF
• P16/CDK1 block cyclin D/CDK2 phosphorylation of RBat the G1/S checkpoint
• P14/ARF activates p53 by inhibiting MDM2
!
The TGF-B Pathway
• TGF-B is an important inhibitor of proliferation
• TGF-B binds to its receptor leading to dimerization and activation of receptor SMADs
• These then enter the nucleus, bind to SMAD4, and upregulate transcription of CDKIs
• The same signaling leads to repression of transcription of c-myc, CDKs, and cyclins required
for RB inactivation (forces a pause at the G1/S checkpoint)
• This pathway is mutated in100% of pancreatic cancers
!
PTEN
• A tumor suppressor gene located in chromosome 10
• PTEN inhibits activation of the PI3K/AKT pathway
• AKT phosphorylates MDM2 which enhances destruction of p53
• AKT also inactivates the TSC1/TSC2 complex releasing mTOR, which stimulates uptake of
nutrients needed for growth and augments the activity of several factors required for protein
synthesis
• The PI3K/AKT pathway is probably the most commonly mutated pathway in human cancer
!
NF1
• A tumor suppressor gene encoding neurofibromin on chromosome 17
• Neurofibromin is a GAP that influences signaling through the RAS pathway
!
NF2
• Encodes merlin on chromosome 22
• Merlin shows homology with cytoskeleton associated proteins
• Cells lacking merlin are not capable of establishing cell-cell junctions and are insensitive to
normal growth inhibitory signals generated by cell-cell contact
!
VHL
• The VHL protein on chromosome 3 is part of a ubiquitin ligase complex
• In the presence of oxygen, HIF1 is bound by VHL protein leading to ubiquitination and
destruction
• In hypoxic environments, HIF1 is free to enter the nucleus and activates many genes related
to angiogenesis
!
WT1
• WT1 is located in chromosome 11 and regulated mesechymal to epithelial transition in renal
development
• Loss of WT1 likely prevents differentiation
• The mechanisms of WT1 in tumors are poorly understood
!
Patched (PTCH)
• Tumor suppressor genes encoding a cell membrane protein that functions as a receptor for
Hedgehog
• Regulates several genes including TGF-B, and PDGFR a and b
• Are present in 20-50% of sporadic cases of basal cell carcinomas
!!
EVASION OF APOPTOSIS
The 2 main pathways of induction of apoptosis and extrinsic and intrinsic:
!
Extrinsic Pathway:
• Signalling through the death receptor CD95/Fas
• Initiated when CD95 binds to the Fas ligand and trimerizes its cytoplasmic death domains
which activates FADD
• FADD cleaves and activates procaspase 8 which then activates downstream caspases
• Caspase 3 cleaves that DNA at specific sites resulting in cell death
• Caspase 8 can also activate BID which activates the intrinsic pathway as well
!
Intrinsic Pathway:
• Signaling due to DNA damage that cannot be repaired
• Results in permeabilization of the mitochondrial membrane with release of cytochrome c
that initiates apoptosis by activating caspase 9
• Caspase 9 can activate caspase 3
• Pro-apoptoptic proteins BAX and BAK are required for apoptosis and directly promote
mitochondrial permeabilization
!
• The action of pro-apoptotic proteins is inhibited by anti-apoptotic proteins (eg. BCL2).
• A third set of proteins (PUMA, BAD, BID) regulate the balance between the pro and anti-
apoptotic proteins.
• The caspases can be inhibited by a family of proteins called inhibitor of apoptosis proteins (IAPs).
Some tumors avoid apoptosis by upregulating IAPs.
• Mutations at various points of this pathway can lead to evasion of apoptosis.
!

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LIMITLESS REPLICATIVE POTENTIAL: TELOMERASE
• Most normal human cells have a capacity of 60 – 70 doublings before they lose their ability
to divide and become senescent
• The mechanism for this is hypothesized to be due to progressive shortening of telomeres
• Short telomeres are recognized by DNA repair machinery as double stranded DNA breaks –
leads to cell cycle arrest through actions of RB and p53
• If the checkpoints are disabled, the non-homologous repair pathway will fuse the shortened
ends of the chromosomes together resulting in dicentromeric chromosomes that are pulled
apart at anaphase resulting in new double stranded breaks
• Mitotic catastrophe and massive cell death eventually occurs
• Tumor cells must develop strategies to avoid mitotic catastrophe
• If the cell manages to reactivate telomerase it may escape death
• The period of instability prior to telomerase activation allows numerous mutations to
accumulate
• 85 – 95% of cancers express telomerase
!
!
ANGIOGENESIS
• Tumors cannot enlarge beyond 1 – 2mm unless they are vascularized
• Tumor vasculature is abnormal – leaky and poorly organized
• Angiogenic factors may be produced by tumor cells themselves, associated inflammatory
cells, or stromal cells
• Many proteases released from the tumor stroma or parenchyma are proangiogenic
• Tumor hypoxia induces HIF which promotes angiogenesis
• P53 can stimulate expression of anti-angiogenic factors, therefore loss of p53 provides a
more permissive environment for angiogenesis
• Bevacizumab is an anti-VEGF drug used to treat cancer
!
!
INVASION AND METASTASIS
• Although millions of tumor cells are released into the blood each day from a primary tumor,
only a few metastasis are produced
• The metastatic cascade involves 2 main phases:
• Invasion of the ECM
• Vascular dissemination, homing of tumor cells, and colonization
!
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THE METASTATIC CASCADE
!
Invasion of Extracellular Matrix
• Invasion of the ECM involves 4 steps;
1. Dissociation of cells from each other
2. Degradation of the ECM – overexpression of collagenases and MMPs
• A second mode of invasion is “ameboid migration” where the
tumor cells squeeze through the BM and travel quickly along
collagen fibers
3. Attachment to new ECM components
• Loss of polarization and attachment to the BM normally induces
apoptosis
4. Migration of tumor cells
!
Vascular Dissemination and Homing of Tumor Cells
• In the circulation, tumor cells can be destroyed by a variety of mechanisms
(shear stress, anoikis – apoptosis stimulated by loss of adhesion to the BM,
innate and adaptive immunity)
 • Formation of platelet tumor aggregates favors survival and implantability
• Tumor cells can also bind and activate coagulation factors – emboli
• CD44 is a receptor used by T lymphocytes to bind to endothelial cells and leave the
circulation – overexpression of CD44 may favor mets
• Most tumor mets occur in the first capillary bed the tumor encounter, however, many
cancers do not metastasize along natural patterns of drainage
• Organ tropism may be related to the following:
• Different capillary beds express different adhesion ligands for adhesion molecules
and tumors may express different adhesion receptors
• Some tumors (eg. breast) express certain chemokines attracting them to specific sites
• In some cases, the first capillary bed may be a nonpermissive environment for the
met to grow (tumors rarely go to skeletal muscle despite extensive vascularity)
• Some micromets are thought to become dormant resulting in prolonged survival of
micromets without tumor progression
• Tumor cells are capable of secreting cytokines, growth factors, and ECM molecules that help
to make the metastatic site habitable
• Breast ca secretes PTHrP which stimulate RANKL in osteoblasts, ultimately
activating osteoclasts to degrade bone and release growth factors from the bone
ECM – this results in a favorable sit for a met and a lytic bone lesion

!
!
!
Molecular Genetics of Metastasis Development
Four schools of thought:
1. As mutations accumulated in cancer cells the tumor population becomes heterogeneous
• Eventually, a clone will acquire all the mutations needed to metastasize
2. Breast cancers that have not metastasized have similar expression profiles as the metastasis
• Potential for metastasis may develop early in the development of a tumor
• However, gene expression analysis would not identify a small subclone in the larger
tumor
3. Background genetic variation may contribute to the generation of mets
• In mice, cancer induced with the same mutations have different outcomes in
different strains
4. If tumors derive from tumor stem cells, then mets require that the tumor stem cells acquire
the ability to metastasize
• Recent work has shown that certain miRNAs can may either promote or suppress
metastasis
• Epithelial mesenchymal transition has been shown to favor a promigratory
phenotype in breast cancer (downregulation of epithelial markers such as e-cad and
upregulation of mesenchymal markers such as vimentin) – has only been
documented in breast ca
!
!
GENOMIC INSTABILITY – ENABLER OF MALIGNANCY
What are the 3 major DNA repair systems that are dysfunctional in cancer?
1. Mismatch repair - proofreaders
2. Nucleotide excision repair
3. Recombination repair
!
Hereditary Nonpolyposis Colon Cancer Syndrome (HNPCC)
• Results from defects in DNA mismatch repair – results in gradual accumulation of errors in
the DNA
• Microsatellite instability is the hallmark of MMR defects and is seen in HNPCC and sporadic
colon ca
!
Xeroderma Pigmentosum
• Results from defects in the nucleotide excision repair system – required to repair DNA
damaged by radiation exposure
!
Diseases with Defects in DNA Repair by Homologous Recombination
• Bloom syndrome, ataxia-telangiectasia, fanconi anemia are examples
• BRCA1 and 2 associated with many members of the homologous protein repair pathway to
repair intra and inter strand DNA crosslink’s
• Failure to repair the crosslink’s leads to double strand breaks, activation of the salvage
nonhomologous end-joining pathway, formation of dicentric chromosomes and massive
aneuploidy
• BRCA1 and 2 are rarely inactivated in sporadic breast ca in contrast to APC and p53
!
!
STROMAL MICROENVIRONMENT AND CARICNOGENESIS
Name 3 ways that the tumor stroma contributes to tumor growth:
• The relationship between the tumor parenchymal and stromal cells is complex
1. The ECM stores multiple growth factors and proteases that can be used by the tumor
2. There are also growth promoting relationships between the inflammatory cells,
fibroblasts, and tumor parenchyma
• The desmoplastic stroma is not simply and response to contain the tumor – it is
part of the tumor and may promote invasion and mets
 3. Some predictions of tumor behavior based of gene expression profiling are turning out
to be based on genes expressed by the stroma, not the tumor
!
!
METABOLIC ALTERATIONS: THE WARBURG EFFECT
What is the Warburg effect?
• Even in the presence of ample oxygen, cancer cells can choose to generate ATP via
glycolysis – termed the Warburg effect (aerobic glycolysis)
• This metabolic alteration is very common in tumors and it used clinically to visualize tumors
by PET scanning
• The significance of this alteration is not entirely clear
• Altered metabolism may give a growth advantage in the relatively hypoxic tumor
microenvironment
• Tumor blood vessels are abnormal and the environment remains hypoxic as a result
• HIF1 increases both angiogenesis and proteins required for glycolosis
• By decreasing oxygen demand of individual tumor cells, the oxygen supply is
increased allowing the tumor to grow larger
• The Warburg effect refers to glycolysis that occurs even when there is adequate oxygen for
aerobic metabolism – the changes that promote glycolysis in tumor cells must become fixed
at some point
• Continuous rounds of tumor hypoxia may select clones that are fixed in glycolysis
• Certain mutations in oncogenes and tumor suppressors may favor glycolysis
• Tumor cells need to double all of their membrane component and organelles in
order to divide as well – if glucose is metabolized by glycolysis, the pyruvate
generated can be shunted to anabolic pathways to accomplish this
!
What is autophagy?
• An adaptive response of normal cells to glucose and oxygen deprivation where cells can use
their own organelles for energy sources
• Tumor cells seem to grow without triggering autophagy
• Autophagy may be one method that tumor cells use to become dormant
!
!
DYSREGULATION OF CANCER ASSOCIATED GENES
!
Chromosomal Changes
What 2 types of chromosomal changes can activate proto-oncogenes?
• Translocations
• Inversions
• Reduplication and amplification
• 2 patterns of amplification are double minutes and homogeneous staining regions
(HSRs)
• Double minutes are extrachromosomal segments of DNA that are small and
commonly circular
• HSRs result from insertion of the gene into a new location with subsequent
amplification – lack the normal g-banding pattern
!
Deletions
What chromosomal change tends to be associated with loss of tumor suppressor genes?
• Deletions
!
Epigenetic Changes
• Reversible, heritable changes in gene expression that occur without mutation
• Modification of histones and DNA methylation are examples
 • Cancer cells are characterized by global hypomethylation and selective promoter
hypermethylation (mostly of tumor suppressors)
!!
MiRNAs and Cancer
• Involved in post transcriptional gene silencing
• Can contribute to neoplasia by increasing expression of oncogenes and reducing expression
of tumor suppressor genes
• Oncomires – oncogenic miRNAs

!!
Molecular Basis of Multistep Carcinogenesis
• Cancer must result from the accumulation of multiple mutations
• Adenoma-carcinoma sequence in colorectal cancer
o Inactivation of APC
o Activation of RAS
o Loss of a tumor suppressor gene on 18q, loss of p53
• The majority of cells in colon adenomas are senescence, the loss of p53 allows cancer cells
to escape senescence

!
Carcinogenic Agents and their Cellular Interactions
!
Steps Involved in Chemical Carcinogenesis
• Initiation – initial exposure to a carcinogen causing permanent DNA damage
o Highly reactive electrophiles, targets are DNA, RNA, proteins
o Direct and indirect acting
• Promoters – induce cancer in initiated cells, usually non-tumorigenic by themselves,
generally do not effect the DNA directly and produce reversible cellular changes that
enhance the proliferation of initiated cells

!
Direct Acting Agents:
• Require no metabolic conversion to become carcinogenic
• Some chemo agents are in this category and the risk is induction of a cancer due to the
chemo after treatment of the initial cancer
!
Indirect Acting Agents:
• Require metabolic conversion
• Present in cigarette smoke and hydrocarbon fuels
• Susceptibility is partly determined by polymorphisms in the p450 system from person to
person
• Age sex and nutritional status are also important variables
!
• Aflatoxin B (from Aspergillus):
• produces mutations in the p53 gene (characteristic GC – AT transversion in codon 249)
• In areas where food contamination with Aspergillis is high, this signature p53 mutation
can establish aflatoxin as the causative agent of HCC. This mutation is rare in liver
tumors occurring in areas where aflatoxin contamination of food is not a risk factor
!
!
!
RADIATION CARCINOGENESIS
!
Ultraviolet Rays
• UVB light causes pyrimidine crosslink’s to form in the DNA that are repaired by the nucleotide
excision repair pathway
• Excessive sun exposure may overwhelm the ability of the nucleotide repair system,
forcing the cell to initiate salvage repair mechanisms that favor survival at the expense of
accumulating mutations
!
Ionizing radiation
• Most frequent cancers are AML and CML
• Thyroid cancer closely follows in young people
• Solid cancers of breast, lung, and salivary glands follow
• Skin, bone, and GI tract are relatively resistant, however, any cell can be transformed by
sufficient exposure to radiant energy
!
!
MICROBIAL CARCINOGENESIS
!
Oncogenic RNA Viruses
Human T-Cell Leukemia Virus Type 1 (HTLV1)
• An RNA retrovirus that causes endemic leukemia in Japan and parts of the Carribean basin
• HTLV-1 has tropism for CD4 T cells (the major target for neoplastic transformation
• Leukemia develops in only 3 – 5% of infected individuals after a long latency (50 years)
• One of the viral proteins (tax) is a transcription factor that increases transcription of FOS and
IL-2, and inactivates p16, and enhances cyclin D activation, and activate NF-kB (pro-survival
influence)
!!
Oncogenic DNA Viruses
Name 3 oncogenic DNA viruses:
1. HPV
2. EBV
3. HepB/C
!
Human Papillomavirus
• High risk HPV involved in carcinoma of the cervix and anogenital region
• In benign HPV lesions the viral DNA is maintained in an episome while in cancers, the viral
DNA is integrated into the host genome
• Host integration disrupts the viral DNA leading to loss of the E2 viral repressor and over-
expression of E6 and E7
• E7 displaces E2F from RB promoting progression
through the G1/S checkpoint, also inactivates
p21 and p27 and activates cyclin E and A
• E6 binds to and mediates degradation of p53 and
BAX and activates telomerase
• Human p53 is polymorphic at amino acid 72 –
the ARG variant is more susceptible to
degradation by E6 and women with this variant
are more susceptible to developing cervical ca
• Additional mutations are required for invasive
SCC to occur
!
!
Epstein-Barr Virus
• Member of herpes family
!
Name 7 tumors where EBV is involved in the pathogenesis:
1. African Burkitt lymphoma
2. B-ell lymphomas in immunosuppressed
3. Subset of Hodgkin lymphoma
4. Nasopharyngeal carcinoma
5. Some gastric carcinomas
6. Some rare T cell lymphomas
7. NK cell lymphomas
!
How does EBV infection lead to neoplasia?
• EBV enters B cells by binding the CD21 receptor
• B cells are latently infected and become immortalized
• One of the EBV proteins (LMP-1) behaves as a constitutively active CD40 receptor
(stimulates B cell growth)
• LMP-1 also activates NK-kB and JAK/STAT pathways
• LMP-1 also activates Bcl-2 to prevent apoptosis
• EBNA-2 mimics a constitutively active Notch receptor and activates cyclin D and src proto-
oncogenes
• The EBV genome also contains a viral IL-10 (stolen from the host genome) that when
expressed inhibits macrophages from activating anti-viral T-cells
• Immunologically normal people can manage the infection
!
Burkitt lymphoma:
How do you classify Burkitt lymphoma?
1. Endemic (African)
2. Sporadic
3. Immuno-suppression related
!
Additional factors other than EBV infection are required for the
development of Burkitt lymphoma:
• EBV infection is ubiquitous
• EBV is positive in only 15–20% of sporadic Burkitt
• diffs b/w non-neoplastic EBV transformed B cells and BL cell
line (Burkitt cell lines do not express LMP-1 and EBNA-2)
• likely in endemic areas, other infections such as malaria,
impair immunity allowing sustained B-cell proliferation
• EBV infected cells have time to adapt by down regulating viral
proteins targeted by T-cells and persist even when normal
immunity returns
• If these persistent EBV transformed cells acquire specific
mutations (activation of c-myc), Burkitt lymphoma may
result
• Tumors lacking EBV contain translocations (8:14) that lead
to c-myc dysregulation
!
Immunosuppresion related B cell lymphomas:
• These tumors uniformly express LMP-1 and EBNA-2
• These tumors generally regress when the immunosuppression is
reversed
Nasopharygeal Carcinoma:
• 100% of NPC from all parts of the world contain EBV
• geographic distribution suggests that other environmental or genetic factors may be
important
Hepatitis B and C Viruses
• 70 – 85% of HCC worldwide are associated with HBV or HCV
• HBV is endemic in the far east and Africa
• Most of action in carcinogenesis is incompletely understood
• HBV and C genomes do not encode any oncoproteins
• Integration of HBV is random
• The dominant oncogenic effect appears to be mediated by chronic inflammation induced by
the viral infection
• In the setting of unresolved, chronic inflammation, the immune response becomes
maladaptive and potentially carcinogenic
!
Helicobacter Pylori
• HP is the first bacterium described as a carcinogen
• Involved in the genesis of gastric carcinomas and lymphomas
• Development of carcinomas is thought to be due to unresolved chronic inflammation –
occurs over decades in 3% of infected patients
• HP strains associated with gastric carcinoma contain a “pathogenicity island” that contains
cytotoxin associated A gene
• CagA has a variety of effects including unregulated growth stimulation
• Strain specific HP is also implicated in MALT lymphoma
• Chronic infection and host and strain specific factors favor neoplasia
• In early stages, the monoclonal MALToma relies on T cell stimulation for NF-kB expression
• In later stages, the MALToma may acquire mutations (such as t11:18) giving constitutively
active Nf-kB in the absence of T-cell signaling
!!
HOST DEFENSE AGAINST TUMORS – TUMOR IMMUNITY
!
• Paul Ehrlich first proposed that immune cells may be capable of eliminating tumors
• Define immune surveillance:
• A normal function of the immune system is to survey the body for emerging malignant
cells and destroy them
• Cancer immunoediting refers to the effects of the immune system in preventing tumor function
and also to the selection of clones and escape immune detection.
!
TUMOR ANTIGENS
• Tumor-specific antigens: expressed only on tumor cells
• Tumor-associated antigens: present on tumor and normal cells
!
Give 6 examples of tumor antigens:
! 1. Products of mutated genes
• May be recognized by class I or II MHC pathways and activate corresponding CD8
T-cells of CD4 T-cells
• These altered proteins are not present in normal cells so should not induce tolerance
! 2. Overexpressed or aberrantly expressed cellular proteins
• Some antigens are produced in very low levels in normal cells
• When massively overproduced in cancers, they may fail to induce tolerance
• Cancer-testis antigens are encoded by genes only expressed in the immune
privileged testis and therefore act tumor specific
! 3. Tumor antigens expressed by oncogenic viruses
• The competent immune system plays a major role in surveillance against viral
induced tumors (HPV vaccines)
!
 4. Altered cell surface glycolipids and glycoproteins
• Several mucins have been the focus of diagnostic and therapeutic studies including
CA-125 and CA-19-9 and MUC-1
• MUC-1 is an integral membrane protein that is normal only expressed on the apical
surface of breast ductal epithelium – sequestered from the immune system
! 5. Cell type-specific differentiation antigens
• Molecules normally present on the cell of origin of the tumor
• Do not induce an immune response, however they are targets of immunotherapy
(eg. CD20 in B cell tumors)
• Idiotypic determinants of the surface immunoglobulin are specific for that clonal
population because all other B cells produce different idiotypes
! 6. Oncofetal antigens
• Expressed at high levels in fetal cells and cancer cells but not in normal tissues –
silenced during development and derepressed in malignancy
• These proteins are also elevated in many inflammatory conditions
• No evidence that these are targets of antitumor immunity
• Main clinical utility is tumor detection at diagnosis
!
!
ANTITUMOR EFFECTOR MECHANISMS
What is the dominant anti-tumor mechanism in vivo?
• Cell-mediated immunity
!
Describe 4 different anti-tumor immune mechanisms:
1. Cytotoxic T-lymphocytes
• Well described especially against virally induced tumors
2. NK cells
• Capable of destroying tumor cells without prior sensitization
• Tumors that fail to express MHCI cannot be recognized by T cells but may trigger NK
cells
• NK cells can recognize stress induced antigens on tumor cells
3. Macrophages
• Exhibit cytotoxicity against tumor cells in vitro
• Activated by T cells and NK cells
4. Antibodies
• No evidence that antitumor antibodies form spontaneously but administration of
monoclonal antibodies against tumor cells can be therapeutically effective (eg.
Monoclonal antibodies against CD20 in B-cell lymphoma)
!
!
IMMUNE SURVEILLANCE AND ESCAPE
• Strongest argument for the existence of immune surveillance is the increased frequency of cancer
in immunodeficient hosts.
• Since most cancers develop in immunocompetent hosts tumor cells must evade the immune
system. Name 6 ways that tumors can evade the immune system:
1. Selective outgrowth of antigen negative variants
2. Loss or reduced expression of MHC
• Cells without MHC class I cannot be recognized by T cells
3. Lack of costimulation
• Sensitization of T cells requires 2 signals
• Tumor cells may express antigens on MHC class I but may lack
costimulatory signals required for T cell activation
• Without costimulatory signals T cells may become anergic or apoptotic
 4. Immunosuppression
• TGF-b secreted in large quantities by tumors is a potent immunosuppressant
• Some tumors activate regulatory T cells that suppress the immune system
5. Antigen masking
• Tumor antigens may be masked by excessive glycocaylx molecules
6. Apoptosis of cytotoxic T-cells
• Some cancers express FasL and may induce apoptosis in Fas-expressing T
lymphocytes
!
• also some recent evidence that paradoxically, the immune system may promote tumor growth by
providing growth factors and suppressing the host response to the tumor.
!
Clinical Aspects of Neoplasia
• Name 5 major clinical consequences of neoplasia:
1. Location and impingement on normal structures
• Small benign tumors can have serious effects in a bad location (eg. Pituitary tumor)
2. Functional activity (hormone synthesis or paraneoplastic syndromes)
• Beta-cell pancreas adenomas can cause life threatening hypoglycemia
3. Bleeding and infections when the tumor ulcerates
4. Symptoms from rupture or infarction
5. Cachexia/wasting
• Weight loss in cachexia results equally from loss of fat and muscle
• BMR is increased even though food intake is decreased
• TNF may mediate cachexia – at high concentrations it suppresses appetite and
mobilizes fat stores, other cytokines likely contribute
• Cachexia hampers chemo because reduced doses must be given
• 1/3 of cancer deaths are attributed to cachexia rather than tumor burden
!
Paraneoplastic Syndromes
• Definition – symptom complexes in patients with cancer that cannot be readily explained, either
by local or distant spread of the tumor, or by the elaboration of hormones indigenous to the
tissue from which the tumor arose
• May be the presenting feature of the tumor, may be lethal, and may mimic metastatic disease.
!
• Classify paraneoplastic syndromes:
1. Endocrinopathies
• Cushing syndrome, SIADH, hypercalcemia, carcinoid syndrome, polycythemia
2. Nerve and muscle syndromes
• Myasthenia
3. Dermatologic disorders
• Dermatomyositis, acanthosis nigricans
4. Osseous, articular, and soft tissue changes
• Clubbing
5. Vascular and hematologic changes
• Venous thrombosis, marantic endocarditis, DIC (acute promyelocytic leukemia)
6. Other
• Nephritic syndrome
!
• Hypercalcemia is the most common paraneoplastic syndrome. Overtly symptomatic
hypercalcemia is more likely to be related to cancer than hyperparathyroidism.
• Can be secondary to osteolysis from primary bone tumors or mets (not a paraneoplastic
syndrome)
• Can be secondary to the production of calcemic humoral substances from extra-osseous
neoplasms (a paraneoplastic syndrome)
• PTHRH is produced in small amounts by many normal tissues (skin, muscles, bone,
ovary)
 • Regulates Ca transport across the lactating breast and placenta and regulates
development and remodelling in the lung
• What is the most common lung neoplasm associated with hypercalcemia?
• Squamous cell bronchogenic carcinoma
!!
!
GRADING AND STAGING OF TUMORS
• Grading is based on the degree of differentiation of tumor cells. Mitotic counts and architectural
features may also be incorporated into grading schemes.
• Staging is based on the size and spread of tumors including regional lymph nodes and distant
mets.
!
!
LABORATORY DIAGNOSIS OF CANCER
Histologic and Cytologic Methods
• Specimens must be adequate, representative, and properly preserved
!
Immunohistochemistry
• Give 3 examples of how immunohistochemistry can be used in the diagnosis or management of
malignant tumors:
1. Categorization of undifferentiated malignant tumors
2. Determination of sit of origin of metastatic tumors
3. Detection of molecules that have prognostic or therapeutic significance
!
Flow Cytometry:
• Name 2 broad uses of flow cytometry:
1. Determination of DNA content of cells
2. Analysis of membrane antigens to demonstrate clonality
!
Molecular Diagnosis:
• List 4 broad uses of molecular diagnoses in cancer:
1. Diagnosis of malignant neoplasms
• Detection of translocations by PCR
• CHG and microarrays are being increasingly used to characterize tumors
2. Prognosis of malignant neoplasms
• N-myc amplification is poor prognosis in neuroblastoma
• Confirmation of Her2 amplification
• Loss of 1p and 19q in oligodendroglioma
3. Detection of minimal residual disease
4. Diagnosis of hereditary predisposition to cancer
!
Molecular Profiles of Tumors
• The most commonly used tool for large scale analysis of gene expression is DNA microarray
technology
• Laser capture microdissection can be used to selectively capture tumor cells
• In breast cancer, a 70 gene prognosis signature has been established
• A smaller 21 gene panel (Oncotype Dx) is currently being used to assess the risk of recurrence
and likely benefit of chemo for a subset of breast cancer patients
!
Tumor Markers
Name 6 classes of tumor markers with examples:
1. Hormones
• HCG, Calcitonin, Catecholamines and metabolites
2. Oncofetal antigens
 • AFP, CEA
3. Isoenzymes
• PAP (prostatic acid phosphatase), NSE
4. Specific proteins
• Immunoglobulins, PSA
5. Mucins and glycoproteins
• CA-125, CA-19-9
6. Molecular markers
• P53, APC, RAS mutants

Paraneoplastic Syndromes
Clinical Syndromes Major Forms of Underlying Cancer Causal Mechanism

ENDOCRINOPATHIES
Small cell ca of lung
Cushing Syndrome Pancreatic ca ACTH or ACTH-like substance
Neural tumors
Small cell ca of lung; intracranial
SIADH secretion ADH or ANP hormones
neoplasms

SqCC of lung
Breast ca
Hypercalcemia PTH-RP, TGFα, TNF, IL-1
Renal cell ca
Adult T-cell leukemia/lymphoma
Ovarian ca
Hypoglycemia Fibrosarcoma
Insulin or insulin-like substance
Other mesenchymal sarcomas
HCC
Carcinoid syndrome Bronchial carcinoid
Serotonin, bradykinin
Pancreatic ca

Gastric ca

Polycythemia Renal cell ca

NERVE AND MUSCLE SYNDROMES

Myasthenia Bornchogenic ca Immunological

Disorders of CNS and PNS Breast ca

DERMATOLOGIC DISORDERS

Gastric ca
Acanthosis nigricans Lung ca Immunological, EGF secretion
Uterine ca
Dermatomyositis Bronchogenic, breast ca Immunological

OSSEOUS, ARTICULAR, ADN SOFT TISSUE CHANGES

Hypertrophic osteoarthropathy and

VASCULAR AND HEMATOLOIGIC CHANGES

Pancreatic ca
Venous thrombosis (Trousseau
Bronchogenic ca Mucins that activate clotting
syndrome)
Others
Non bacterial thrombotic
Advanced cancers Hypercoagulability
endocarditis

Red cell aplasia Thymic ca Unknown

OTHERS