Molecular Biology and Diagnostics

MOLECULAR ONCOLOGY AND APPROACH TO DIAGNOSIS

• Types:
HALLMARKS OF CANCER o Viral Oncogenes (v-oncs)
All these 6 points need to be present, in any order, for a cell to o Cellular Oncogenes (c-oncs)
become malignant:
▪ Self-sufficiency in growth signals B. TUMOR SUPPRESSOR (TS) GENES
▪ Insensitivity in anti-growth signals • Inhibit growth and multiplication of mutated cells
▪ Tissue invasion and metastasis • Prevent neoplastic transformation
▪ Limitless replicative potential • Recessive and highly conserved
▪ Sustained angiogenesis o Always on the “on” state.
▪ Evading apoptosis o Recessive – need to destroy both/pair of TS gene to lose
function. (will still somewhat function even if only 1
chromosome of the pair is working)

• CLASSIFICATION OF TS
o Cell Adhesion Molecule – APC, DCC
o Regulators of the Cell Cycle – RB1, Tp53

• KNUDSON’S TWO-HIT HYPOTHESIS

o 1st hit: TS mutation or inherited mutation
o 2nd hit: gross chromosomal loss

• TYPES OF TS GENE DEFECTS

o Wild-type TS gene
▪ Occurs in the natural state
▪ Sporadic mutation
Figure 1. Hallmarks of Cancer ▪ 1st hit: new TS gene mutation in 1 of the Chr pair
▪ 2nd hit: damage/deletion of normal TS gene in the other pair
CANCER CHARACTERISTICS
• Clonality
• Autonomy
• Anaplasia
• Metastasis

CLONALITY
• Genetic disease at the cellular level
• Mutations play a critical role in its pathogenesis
• Consequences of genetic instability:
o Phenotypic Heterogeneity Figure 3. Wild-type TS gene
▪ Mutated parent genes proliferate → different expression of
o Heritable cancer syndromes
the mutation in offspring
▪ Familial type
▪ Note: 5 patients with same malignancy, same age, same
▪ 1st hit: inherited mutation
stage of disease, but some may survive while some won’t.
▪ 2nd hit: damage/deletion of normal TS gene in the other pair
(Individuality of patients!)
▪ Tendency to develop malignancy at an earlier age compared to
o Tumor Progression
wild-type mutations because 1st hit is already present at birth
▪ A proliferating mass of cells → tumor formation
(so only 1 more hit needed to express malignancy phenotype)
• TYPES OF CANCER GENES ▪ Heterozygous TS gene at birth (1 normal, 1 mutated TS gene)
1. Oncogenes → the remaining normal gene becomes damaged/deleted →
2. Tumor Suppressor Genes “loss of heterozygosity” (malignant phenotype)
3. DNA Repair Genes

A. ONCOGENES
• Promote/Accelerate cell proliferation (normal cells: regulate cell
growth)
• Represent the abnormal stimulation of a particular gene.
• Dominant and highly conserved
o When activated, their expression is dominant = only 1 of a pair
of oncogenes needed to be activated for it to express
malignancy potential
Figure 4. Heritable cancer syndrome
• “Off” state of oncogene = proto-oncogene
o Proto-onc → mutation → oncogene

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C. DNA REPAIR GENES
• Ensures fidelity of replication
• Checks and corrects mismatched pairs
• If non-functional:
o Mutation → inefficient repair and replication → increased
propensity of oncogenes and TS genes to undergo mutation
(Microsatellite Instability/MIN)
o MIN – areas of unstable DNA because mutations are already
present there.
o Example of DNA repair gene failure: Hereditary Non-polyposis
Colon Cancer (HNPCC)
• Apoptosis – programmed cell death; as cells grow older, their
tendency to develop mutations gets higher, thus apoptosis is
necessary to prevent continuous proliferation of cells.
Figure 6. When cells age, they develop DNA damage. So before they undergo
mutation, they commit suicide (apoptosis) so no mutations will be passed on
to the next generation.
▪ activates cascade resulting in DNA fragmentation and
apoptosis.
F. TELOMERES AND TELOMERASE
• Telomeres
o Prevent recombination and shortening of the lagging strand
o Repeats of 6 nucleotide sequences
▪ Humans: TTAGGG
o Biologic/Reproductive clock of a cell – the more times a cell
replicates, the higher the chance for it to develop a mutation.
Therefore, cells have telomeres (biologic/repro clock) to regulate
amount of replications that can occur. (When their time is up –
bio/repro clock -- cells commit suicide)
o Replicative Senescence:
▪ Telomeres become shorter in every replication → very short →
sensed by p53 as damaged DNA → apoptosis
o Can be regenerated by Telomerase. (expressed in cancer cells)
• Telomerase
o Adds 6 nucleotide repeats to 3’-OH end of DNA
▪ RNA serves as template
▪ Reverse transcriptase
o Enzyme that maintains telomeres
o Expressed in normal cells during embryologic development d/t
the need for continuous reproduction to form the entire human
body. Switched “off” afterwards except for stem cells.

D. CYCLIN REGULATORS
REVIEW:
• The mechanism of apoptosis is mediated by 2 important TS genes: Oncogene TS Gene DNA Repair
o p21 – inhibits cell cycle progression & permits DNA repair to take Gain of function→ Loss of function→ Loss of function→
place cancer cancer cancer
o p53 – “guardian of the genome” Promote cell Inhibit growth and Checks and corrects
▪ most common genetic alteration found in human cancer proliferation multiplication of mismatched pairs
mutated cells
▪ in the presence of DNA damage:
Ex: Her2-neu, Ras, Ex: p53, Rb, APC Ex: BRCA1, BRCA2
- halts cell cycle progression to facilitate DNA repair, if
Myc
damage is repairable (reactivation of capping of the DNA) *** Multiple/accumulated mutations are needed in development of cancer.
- activates apoptosis in cases of severe DNA damage
(apoptotic pathway) G. MUTATIONS
✓ In the presence of mutated DNA, it stops proliferation, allows
DNA to be proofread, repaired and if repair is impossible then it • Change in the normal base-pair sequence
is moved to level of apoptosis. • Commonly used to define DNA sequence changes that alter
protein function.
E. COMPONENTS OF APOPTOTIC PATHWAY
• May affect 2 types of cells leading to 2 types of mutations:
• BCL-2 family of proteins
1. SOMATIC MUTATIONS
o Pivotal decisional checkpoint
o Occur in non-germline tissues
▪ Anti-apoptotic:
o Non-inheritable - damage to mature cells that do not
- BCL-2
participate in the reproductive process.
- BCL-xL
o Most cases of cancer
▪ Pro-apoptotic:
o Several genes are involved and accumulated mutations
- BAX
needed to develop malignancy since there were no
- BAD
mutations present at birth (no 1st hit yet. See Knudson’s 2-hit
- BAK
hypothesis)
- BID
o Sporadic malignancies
o i.e. main mechanism of lymphoid tumors is overexpression of
o Overall rate increases with age (more common in the
BCL-2 (anti-apoptotic = favors continuous proliferation of tumor
elderly)
cells)
o Because they do not replicate, it is not passed to the next
• Cytochrome C
generation.
o Component of mitochondria released in response to apoptotic
o Tend to accumulate over time and begin when an individual
signals.
is at an older age compared from germline mutations so
o Apoptotic signals → Cytochrome C stimulated → activates cells
OLDER AGE when they manifest malignancy. Majority of
that destroy the cell
patients we encounter in the wards who are 50, 60, 70 years
• Caspases (Cysteine-containing ASPartate-specific ProteASES) old tend to have somatic malignancy.
o Initiator Caspases
▪ activated in response to apoptotic signal
o Executioner/Effector Caspases
 2. GERMLINE MUTATIONS
o Heritable – causes cancer family syndrome and Mendelian
cancer syndrome
o Important to detect! Patients at risk for these mutations
would have other family members that are similarly at risk.
(widened scope of responsibility)
o All cells affected in offspring; risk is present at birth
o Damage to cells involved in the reproductive process (Ovum
in females and/or sperm in males)
o Average age at onset of malignancy: young (usually 30s or
40s, sometimes even 20s), earlier onset of disease.
o If we can recognize these individuals, then we can do
screening to catch the malignancy early or have lifestyle
changes in order to prevent/diminish exposure to certain
substances that can promote mutations that give rise to
malignancy.
o Rapid proliferation phase. ANGIOGENESIS; energy, nutrient,
and O2 requirements increase → hypoxia of tumor cell →
stimulus for new blood vessel proliferation
o phenomenon of antigens fuels growth in the log phase
o Sensitivity to chemotherapy is highest when the cells are
proliferating. Hence, cancer cells in the log phase tend to be
most sensitive to the effects of chemotherapy.
3. PLATEAU phase
o Terminal growth phase. Net growth is usually flat or zero.
MIX of proliferating, resting, and dying cells (in order to
conserve nutrients)
o Tumors in the plateau phase tend to be resistant to
chemotherapy and radiotherapy.
o Need to debulk the tumor to allow it to go back to the log
phase → then, institute chemotherapy

H. HEREDITARY CANCER
• Approximately 5-10% of breast, ovarian, and colon cancers are
linked to hereditary risks.
• Clinical Importance:
o Identified high risk individuals may be motivated to change
behavior/lifestyle
o Early recognition of people at risk results in changes in
management
o Studies of high risk families may contributes greatly to
understanding cancer in the general population

• Clinical features of Hereditary Cancer Syndrome:

o 2 or more relatives in a family have been diagnosed with cancer Figure 9. Gompertzian growth of tumor cell
o >1 generation is affected
o Cancer has been diagnosed in a family member <50 years old • Junction between lag and log phases – cells slowly grow and
o Same type of cancer has occurred in several members of the proliferate - earliest time for cancer to be clinically detected.
family
o >1 type of cancer has occurred in 1 person or a clustering of
specific tumor types is present B. CELL REGENERATION CYCLE
o A rare cancer has occurred in one or more members of the
family.
o Certain ethnic groups (Ashkenazi Jews)

AUTONOMY
• Consequence of germline or somatic mutations.
• Common in tissues with rapid turnover
1. Tissues exposed to environmental agents
▪ Epidermal or lining epithelial tissues (majority of
malignancies encountered now are termed CARCINOMA –
malignancy of epithelial tissue)
2. Tissues whose proliferation is hormone dependent
▪ i.e. breast, prostate Figure 10. As cells enter the S-phase (Synthesis phase), they rapidly
• The growth of cancer is expressed in doubling time proliferate. Thus, high S-phase fraction → aggressive tumor → poor
1. mean length of time for division of all tumor cells present prognosis
2. directly proportional to rate of progression and aggressiveness
of tumor • S-phase – only phase of the cell cycle that is susceptible to
manipulation by external agents (i.e. drugs, radiation)
A. GOMPERTZIAN TUMOR GROWTH • Cyclins, CDKs, and CDKis
o Cyclin and CDK – promote cell cycle
• Tumor cell growth is Gompertzian (s-shaped, see Fig 9), with 3 o CDKi – inhibits cell cycle (cyclin regulator)
phases: ▪ Induced by growth inhibitors
1. LAG phase ▪ Inhibited by positive growth factors
o Initial; slow growth phase. LOW number of cells, circulating ▪ Genetic alterations occur with high frequency in some cancers
factors, and nutrients in the tumor bed.
o Most susceptible portion – no blood supply yet, less
nutrition to fuel growth, no metastasis
2. LOG phase
 METASTASIS
A. ANGIOGENESIS IN TUMOR GROWTH AND METASTASIS

Figure 11. Important gate points:

1. Junction between G2 and M– if promoted by cyclin → cell goes into mitosis
2. Junction between G1 and S – MORE IMPORTANT; commonly affected in Figure 13. Angiogenesis
many malignancies. ANGIOGENESIS
• Clinical importance:
• G2/M checkpoint o Tumor vessel number correlates with risk and degree of
o Regulated by cyclin B/cdc2 (mitosis promoting factor or MPF) dissemination
o Activity of cyclin with its substrate results in: o Cytokines that stimulate endothelial cell proliferation also
▪ Chromosome condensation stimulate proliferation of malignant cells
▪ Nuclear membrane breakdown
▪ Spindle formation X. CARCINOGENS
o Therapeutic goal in this phase: prevention of mitosis in the • Occupation related causes
presence of damaged DNA
• Lifestyle related causes – biggest problem as far as malignancy is
• G1/S checkpoint concerned
o Area most often involved in cancer
o Tobacco, Diet , Sexual practices
o Complex mechanism of regulation; involves phosphorylation of
• Multifactorial causes
Rb gene, which results in:
• Viral carcinogens
▪ Activation of several genes needed for S phase progression
▪ Promotes differentiation through association with • Chemical carcinogens
transcription factors • Ionizing radiation
▪ “gate keeper”
- Keeps the entire replication process in check A. OCCUPATION RELATED
- Conjoined with protein E2F (when non-phosphorylated) – no • Barrier protection is very important to prevent these types of
replication can occur cancers as well as to recognize that an substance that an employee
- Phosphorylated → detaches from protein E2F → E2F sends is dealing with is carcinogenic
signals to copy DNA
▪ i.e. Retinoblastoma – caused by Rb gene mutation; very Table 1. Summary of Occupational Carcinogens
malignant and very rapid progression of tumor because there Carcinogen Occupation Type of Cancer
is no gatekeeper to stop E2F from sending signals to copy the Arsenic Mining, pesticide workers Lung, skin, liver
Asbestos Construction workers Lung, mesothelioma
mutated DNA of the cell → continuous replication of mutated
Benzene Petroleum, rubber, chemical Leukemia
gene
workers
Chromium Metal workers, electoplaters Lung
ANAPLASIA Leather dust Shoe manufacturing Nasal, bladder
A. CONSEQUENCES OF AUTONOMY Naphthylamine Chemical, dye, rubber workers Bladder
• continued proliferation results in mass formation Radon Underground mining Lung
• Unregulated growth results in architectural disorganization Soots, tars, oils Coal, gas, petroleum workers Lung, Skin, Liver
(ANAPLASIA) → histologic abnormalities Vinyl chloride Rubber workers, polyvinyl Liver
chloride
• Loss of normal differentiation and function
Wood dust Furniture manufacturing Nasal
o The more poorly differentiated the tumor is, the more anaplastic
it tends to be, the more aggressive the behavior of the tumor is.
B. LIFESTYLE-RELATED
• MICROSCOPIC APPEARANCE OF CANCER CELLS
i. TOBACCO
• Amount of smoking is directly proportional to the development of
cancer: the more one smokes, the higher the risk of developing
malignancy.
o The most carcinogenic substance ever produced by human
beings for commercial consumption
o It contains substances that contains thousands and thousands of
substances that are carcinogenic
o The cancer plague of our generation
o Many of the substances that are associated with occupational
carcinogens are contained in a single cigarette. That is how much
carcinogenic tobacco is.
o Non smokers are still at risk due to secondhand smoke.
• Tobacco-Related Malignancies EFFECTS OF ALCOHOL AND TOBACCO:
o Lung cancer
o Pancreatic cancer
o Bladder cancer
o Renal cancer
o Cervical cancer

ii. ALCOHOL
• 2nd most common carcinogenic substance produced by human
beings for commercial consumption
• Associated with liver cirrhosis and potential liver cancer
Figure 16. Combination of Alcohol and Cigarettes increases risk for Cancer of
iii. DIET- RELATED RISK FACTORS the Esophagus
RISK FACTORS TYPE OF CANCER
Nitrates Gastric D. MEDICATIONS AS CARCINOGENS
Salt Esophageal
• Anti-CA Treatment
Low Vitamins A, C, E
Low consumption of yellow- green veggies o Anti-CA treatment can damage DNA. Their potential to develop
High fat Low calcium Colon Breast malignancies exists. Therefore it is not unusual for those who
Low fiber Broiled or fried food Pancreatic Uterine have been on therapy for malignancies when they were younger
Prostate develop secondary malignancies 10, 15, 20 years after.
Mycotoxins Liver • Aromatase Inhibitors
**If you eat grain, beans, legumes that are poorly stored and are infested
with certain fungi → AFLATOXINS E. SYNTHETIC HORMONES
• Japanese = high rate of gastric CA due to high intake of pickles and
• The use of estrogens in post-menopausal women to alleviate the
preserved food.
post-menopausal symptoms can increase the development of
• The higher the caloric intake, the higher intake of red meat = the breast cancer
higher the chance of developing colon cancer. • And in women given diethylstilbestrol to alleviate the symptoms of
• People in New Zealand who have nothing there but cows and pregnancy like nausea. The problem is that is passed on to the
sheep have increased risk of colonic CA while people in Nigeria who female fetus and that fetus can be at risk for developing
have nothing to eat have low risk for developing colonic CA. endometrial malignancy later on
iv. OVERWEIGHT and EXERCISE F. IONIZING RADIATION
• WEIGHT – not an actual risk factor per se, but once present, can
aggravate any other risk factor present. • Includes electromagnetic rays & particulate matter
• Recommended: BMI = 25 or less and to exercise at least 3 times a • Mechanism: ↑ free radicals & mutations
week for at least 30 minutes. • Pathology: leukemias > thyroid ca > lung & breast ca
• Radio Resistant tissues: bone, skin and the GIT
v. SEXUAL PRACTICES RISK FACTORS • Radiation because it produces free radicals
RISK FACTOR TYPE OF CANCER • The most sensitive is the bone marrow, followed by the thyroid
Sexual promiscuity Cervical gland, followed by the breast and the lungs
Multiple partners ** Notice that it is only Cervical CA → because the
Unsafe se length of the urethra of men protect htem from HPV G. SOURCES OF POTENTIALLY CARCINOGENIC RADIATION
HPV infection- men are the ones that are most affected • Sunlight
Higher prevalence of cervical CA to those women infected with HPV o Most common source
(vs. those who are not). Vaccine is available that can prevent the o High risk for skin cancer and melanoma
onset. Advise vaccination BEFORE sexual contact because it is o Use a skin protective factor of 15 or higher during dangerous
theorized that once you have sexual contact, you have been exposed times of the day
to HPV. • Artificial sources of UV light
• X-rays (radiotherapy)
C. MULTIFACTORIAL FACTORS
o Generates peroxidases (free radicals) which damage the DNA.
• Tobacco + Alcohol → Oral Cavity Cancer • Radio-chemicals
• Tobacco + Asbestos • Nuclear fission
Respiratory Tract
• Tobacco + Mining Cancer, Lung Cancer
• Tobacco + uranium + radium H. VIRAL CARCINOGENESIS
• Combination of cigarettes with alcohol potentiates the • Viral carcinogens are classified into RNA and DNA viruses.
carcinogenic property of both substances. This is because the • Most RNA oncogenic viruses belong to the family of retroviruses
carcinogenic substances in smoke become soluble in the presence that contain reverse transcriptase → mediates transfer of viral
of alcohol therefore it is easily absorbed by mucosal surfaces. RNA into virus specific DNA.
• Effect is synergistic: Smoking alone = 7-8% risk of developing
cancer, Drinking more than 4 bottles alone = 7-8% risk of
developing cancer, Smoking + Drinking more than 4 bottles = 40
times more risk of a non smoker
I. VIRUSES ASSOCIATED WITH HUMAN NEOPLASIA DEVELOPMENT DIAGNOSIS OF CANCER
• Definitive diagnosis by pathological examination
• Pathologic examination may be done by:
o Tissue samples obtained by endoscopy or surgical biopsy or
FNAB (cytology) - biopsy is the cornerstone of diagnosis
▪ FNAB and tissue cytology usually for masses that are hard to
access like lung and liver masses
▪ The more tissue the better analysis

Figure 1. (Left) Histological diagnosis. Infiltrating ductal carcinoma, breast

(Right) Cytological diagnosis. Acute myelogenous leukemia

Remember: HPV – Cervical Cancer, EBV – Nasopharyngeal

Carcinomas and Hepatitis B virus – Hepatocellular Carcinomas. A. ESSENTIAL ELEMENTS IN A PATHOLOGIC REPORT
• A good pathologic report should include a description of the
J. BACTERIAL: Helicobacter pylori
following essential elements
• creates ulcers in GIT-ulcerated areas are areas of continuous o Anatomic origin: breast, lung, colon, etc.
proliferation and repair o Tissue of origin: carcinoma, sarcoma, lymphoma
• chronic repair/proliferation process can eventually get out of o Degree of differentiation: well, moderate, poor
hand and give rise to malignancy ▪ Poorly differentiated is more aggressive (but may have a
• many patients with gastric cancers have history of chronic peptic better respond to treatment)
ulcer disease
• common ulcer sites: esophagus, stomach, small intestine, large B. PATHOLOGIC EVALUATION OF MALIGNANCY
intestine
• Traditionally done with light microscopy with conventional H&E
• the only bacteria associated with risk for developing cancer stains
• Infectious carcinogens --- Viral and lone bacterial cause of • Immunohistochemical stains: for better characterization
malignancy which is H. Pylori
• Electron microscopy
• Molecular genetics and cytogenetic studie
NINE WARNING SIGNS Table 1.Immunohistochemical Stains
• C- Change in bowel of bladder habits IHS TUMOR
• A- A sore that does not heal Leukocyte common antigen Lymphoma
Cytokeratin Carcinoma
o Particularly a part of the body exposed or a sore formed over a
S-100 protein Melanoma
mass
Vimentin Mesenchymal tumors
• U- Unusual bleeding or discharge Prostate specific antigen Prostate
• T- Thickening of a lump in breast or elsewhere Estrogen receptors Breat
• I- Indigestion or difficulty in swallowing--progressive Neuro specific enolase Neuroendocrine carcinomas
• O- Obvious change in a wart or mole—particularly in a part that is ß-HCG Germ cell turmors
always exposed to trauma, e.g. the foot.
• N- Nagging cough or hoarseness
• U- Unexplained anemia ▪ STAINING IS NOT CHEAP! WE NEED TO BE SYSTEMATIC IN
• S- Sudden, unexplained weight loss REQUESTING STAINING EXAMINATIONS!
o Important to recognize because if you catch cancer early, you Table 2. Other Immunohistochemical Markers
can treat in a higher degree Tumor Marker
o Cancer is an unforgiving disease. Every opportunity that we can Breast carcinoma GCDFP-15, ER, PR
catch it early should be taken as a chance to cure or help the Lung adenocarcinoma TTF-1
patient Medullary Thyroid carcinoma TTF-1, Calcitonin
Merkel Cell carcinoma CD 117
FEATURES OF HEREDITARY CANCER SYNDROMES Hepatocellular carcinoma Hep par-1
Prostate cancer PSA, PAP
• Two or more relatives in a family have been diagnosed with CA Cholangiocarcinoma CK 19
• More than 1 generation is affected Mesothelioma Calretinin
• CA has been diagnosed in a family member <50 years of age
• The same type of cancer has occurred in several members of the
family
• More than one type of CA has occurred in one person or
clustering of specific tumor types is present
• A rare CA has occurred in one or more members of the family
• Certain ethnic groups (eg. Ashkenazi Jews)
• CYTOKERATIN PHENOTYPING – useful for identifying the
primary site of tumor if the patient already presents with
metastasis:

C. USEFUL LABORATORIES IN STAGING CANCER

• Serum tumor markers
o Not diagnostic for CA EXCEPT for AFP which is diagnostic for
non-seminomatous germ cell tumors
o Also used to evaluate tumor response and remission status
o Help determine status of illness because if there is high serum
tumor markers at baseline and drops at treatment, then
treatment is effective. If you monitor and markers begin to rise,
then the patient will eventually develop recurrence of
malignancy.
o Not all tumors will have a tumor marker associated
Marker Upper Limit Half-Life Cancer
AFP 25 u/L 3-6 days Testis, liver
B-HCG <1 ng/mL 18-24 hours Testis, GTD
CEA 5 ng/mL Weeks Colorectal, Breast
Lung
CA 15-3 25 U/mL < 2 weeks Breast
CA 19-9 37 U/L ? Pancreas
CA 125 35 U/L 4-5 days Ovary
PSA 2.5-4.0 ng/mL 2-3 days Prostate
Thyroglobulin 10 ng/mL weeks Thyroid

Table 3. Tumor Markers

o Specific site biopsies
May contribute to staging information especially for those which are
ambiguous
o Serum chemistries
Can provide baseline information
In staging, certain blood examinations are necessary such as
functional assessment of end organs.
o Blood Examinations
a. Liver function test =main site of activation and
deactivation of chemotherapeutic agents. Hepatic
insufficiency can lead to prolonged activation or prolonged
deactivation which leads to toxicity.
b. Kidney function test= chief way by which drugs are
excreted. Renal insufficiency can lead to prolonged
retention of drug and toxicity.
✓ In the presence of these insufficiency, there is a
need to modify the therapeutic agents being
used.
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