CANCER GENOMICS

A scale of the cancer problem

Chances to develop invasive cancer
Cancer type

Birth-39

40-59

Birth death

All cancers,

1/69

1/52

1/12 1/24 1/288 1/138 1/48

1/208 1/89 1/115 1/199

1/11

1/2 1/8 1/117 1/37 1/6

1/3

Male/Female

Male/Female

Male/Female

Breast

1/229 Uterine corpus 1/567 Uterine cervix 1/2097 Prostate <1/10000 Lymphoma 1/591 1/1311 Lung 1/3060 1/3099 Colorectal 1/1508 1/1719 Melanoma 1/769 1/508

1/317 1/48 1/116 1/13 1/145 1/17 1/261 1/58

1/57 1/17 1/18 1/82

AGE-ADJUSTED CANCER DEATH RATES

(Males)
Last 70 years statistics
GASTRIC CANCER down 4 times REASON? better nutrition? Probably
LUNG CANCER UP!!! 10 times (due to smoking and air pollution)

AGE-ADJUSTED CANCER DEATH RATES

(Females)
Last 70 years statistics
UTERINE CANCER (including cervix) down 5 times due to early diagnostics
GASTRIC CANCER down 4 times Same as in males. LUNG CANCER UP!!! 10 times (due to smoking and air pollution)

Does science help patients?

5-year survival rates
YES!
Cancer type Leukemia Prostate Breast Years 74-76 35% 68% 75% Years 83-85 42% 76% 79% Year 92-97 46% 97% 87%

NO!
Cancer type Pancreas Liver Uterine corpus Years 74-76 3% 4% 89% Years 83-85 3% 6% 85% Year 92-97 4% 6% 86%

HOW SCIENCE CAN HELP CANCER PATIENTS?

Few things contribute Early diagnosis Specific treatment

Early check for relapse

NOT possible without understanding of the state of genes in every given tumor

Tumors are very polymorphic entities

There is no such a thing like tumor in general; they all differ in their way to survive and kill

Multiple systems of tumor classification exist

ORGAN based (trivial) TISSUE - based (histological) Carcinoma Sarcoma Lymphoma WISDOM based (clinicians experience)

Breast cancer
Brain cancer Liver cancer Colon cancer

Grading/staging (metastase/invasion)

2
Mucinous cystadenocarcinoma Carcinomas with spindle and/or giant cells

Pace of progression (slow of fast)

(best working)

ORGAN based classification

Breast cancer as an example

Tumors can be derived from ductal cells or lobular cells

?
Ductal carcinoma in situ (DCIS) Lobular carcinoma in situ (LCIS)

malignant
High chances to become

benign
Low chances to become

Invasive carcinomas (ductal or lobular or even mixed) Very malignant

inflammatory breast cancer, medullary carcinoma, mucinous carcinoma, Paget's disease of the nipple, Phyllodes tumor, and tubular carcinoma.

TISSUE-based classification
Carcinoma
malignant neoplasm of epithelial origin, including internal epithelium (bladder lining, pancreatic duct lining etc. )

Adenocarcinoma
From tissue/cells producing secret milk or mucous or enzymes

Squamous cell carcinoma (SCC)

From pure lining cells or from skin epithelium

Other small variants, like basal cell carcinoma etc.

Sarcoma
Sarcoma refers to cancer that originates in supportive and connective tissues such as bones, tendons, cartilage, muscle, and fat. Osteosarcoma or osteogenic sarcoma (bone) Chondrosarcoma (cartilage) Leiomyosarcoma (smooth muscle) Rhabdomyosarcoma (skeletal muscle) Mesothelial sarcoma or mesothelioma (membranous lining of body cavities) Fibrosarcoma (fibrous tissue) Angiosarcoma or hemangioendothelioma (blood vessels) Liposarcoma (adipose tissue) Glioma or astrocytoma (neurogenic connective tissue found in the brain) Myxosarcoma (primitive embryonic connective tissue)

Leukemia
cancers of the bone marrow (liquid phase) overproduction of immature lymphocytes, which do not perform as well as they should, therefore the patient is often prone to infection

Myelocytic/myelogenous

erythroblastic

lymphocytic

Mixed (polycytemia vera)

Lymphoma
tumors from the lymphocytes from lymph nodes (solid tumors) Can be present in extranodal (non-lymph nodes) sites stomach lymphoma, skin lymphoma.

Lymphoma goes through the same stages of generalization (metastase) as any solid tumor

Hodgkin disease

Non-Hodgkin lymphoma

Teratoma
Mixed type of tumor, often it is derived from embryonic or stem cells

The most common elements are components of stratified squamous epithelium

medstat.med.utah.edu/

Common types of cancer progression

Adenoma Papilloma Fibroma Lipoma Glioma Adenocarcinoma Squamous cell carcimoma Fibrosarcoma Liposarcoma Anaplastic glioma Glioblastoma multiforme

Chronic leukemia

Remission with latent residual disease Acute leukemic crisis (blast crisis)

WISDOM based classification of tumors

BENIGN MALIGNANT

Borders No invasion

No clear borders Invasion

Normal blood vessels

many leaky blood vessels

newscenter.cancer.gov/sciencebehind/ cancer/cancer34.htm

Histological picture of tumor malignisation

newscenter.cancer.gov/sciencebehind/ cancer/cancer34.htm

TUMOR GRADES are based on degree of cell differentiation

tumor cells obtained from biopsy are evaluated by histopathologist
Grade I (Cells are slightly abnormal and well differentiated) Grade II (Cells are more abnormal and moderately differentiated) Grade III (Cells are very abnormal and poorly differentiated) Grade IV (Cells are immature and undifferentiated)

Tumor histology
Metaplasia
Replacement of normal differentiated tissue by another (differentiated) type like epithelial to mesenchime transition in breast carcinoma progression

Anaplasia
Loss of differentiated phenotype

Dysplasia
Loss of tissue organisation

Hyperplasia
Increase in cell division

TUMOR STAGING
Staging is the classification of the extent of the disease in the body.
Most common systems are TNM system and Numerical system

The tumor, nodes, metastases (TNM) system classifies cancer : -- by Tumor size,

T N

-- the degree of regional spread or Node involvement

-- and presence/absence of distant Metastasis.

Tumor (T)

T0 Tis

(No evidence of tumor)

(Carcinoma in situ limited to surface cells)

T14
(Increasing tumor size and involvement)

Node (N)

N0 No lymph node involvement

N14
Increasing degrees of lymph node involvement

Nx
Lymph node involvement cannot be assessed

Metastases (M)
M0 No evidence of distant metastases M1 Evidence of distant metastases
Pancreatic carc. met. in liver

www.ikp.unibe.ch/lab2/cytostatics/ sld014.htm

Numerical system
Stage 0 Stage 1 Stage 2 Cancer in situ (limited to surface cells) Cancer limited to the tissue of origin with evidence of tumor growth Limited local spread of cancerous cells

Stage 3
Stage 4

Extensive local and regional spread

Distant metastasis

Grading / Staging and prognosis

High-grade tumors progress faster

Late-stage tumors prognosis is worse

newscenter.cancer.gov/sciencebehind/ cancer/cancer34.htm

CANCER TREATMENT
Radical
IDEAL GOAL:

Palliative
GOALS:

Completely eradicate tumor Pain relief

ATTAINABLE GOAL: Infection combat (especially important for leukemias and lymphomas) Chemo-related problems combat

Remove primary tumor (surgically)

or achieve tumor shrinkage (chemotherapy)

RADICAL STRATEGIES of the TUMOR TREATMENT

Radiotherapy
Surgery Chemotherapy

Hormonal therapy
IMMUNOTHERAPY

SURGICAL REMOVAL
Primary care for most of the tumors Ablastic principle (all cuts are done within normal tissue; can be PCR controlled)

On the advanced stages surgery always used in combination with chemotherapy

MINUSES: -Not able to catch and remove micrometastases; -Not able to deal with ascytic (liquid) tumors

CHEMOTHERAPY
CHEMOTHERAPY is the treatment of cancer with drugs that can destroy cancer cells. Ideal anticancer drug should be able to kill tumor cell and be harmless for any normal cell Problem: No clear differences between normal and tumor cells

FIRST PROBLEM WITH CHEMO = side effects

Only the rate of cell division makes tumor cell more prone to poisonous effect of (Cytoreduction) chemotherapy

Normal cells with fast pace of divisions are also very susceptible to chemo

Side effects for BONE MARROW, COLON, HAIR FOLLICLES etc.

SIDE EFFECTS of Chemotherapy

Bone marrow toxicity infection and hemorrhage (bleeding) Gut mucosa toxicity diarrhea

Oral mucosa toxicity mucositis (oral dryness)

Hair follicle toxicity hair loss

Genomics can help!

Better understanding of differences between normal and tumor cells can help to invent new drugs with increased tumor-specific action

SECOND PROBLEM WITH CHEMO = tumor resistance

After rounds of chemotherapy and successful shrinkage of tumor and/or remission tumor cells become resistant to treatment Side effects prevail over benefits

Clinician has to stop treatment and tumor start to grow again

Genomics can help again!

Understanding of genomic changes during development of resistance

can help prevent such changes or help invent new drugs not producing common types of resistance

Traditional chemotherapeutic drugs

I. Alkylating agents (e.g. cisplatin) II. Antimetabolites (e.g. Folate, purine or pyrimidine analogs) III. Plant derivates (vinca alcaloids, taxanes, etoposide) IV. Anti-tumoral Antibiotics
Tumor could become resistant to any of this compounds or their combinations

I. Alkylating agents:
Nitrogen mustards, Nitrosoureas, Platinum agents (Cisplatin), Cyclophosphamides The alkylating agents react with everything; impair cell function by forming covalent bonds with the amino, carboxyl, sulfhydryl, and phosphate groups in biologically important molecules
Alkylating agents generally interact with DNA non-specifically: the more effective ones tend to crosslink DNA.

The electron-rich nitrogen at the N7 position of guanine in DNA is particularly susceptible to alkylation.

Monoalkylation

Alkylating agents interact with DNA non-specifically Active even for the resting cells
(not cell cycle/phase-specific)

Crosslinking (Between strands)

chlorambucil and melphalan

Crosslinking (Same strand)

Cis-platinum

(damage cell now, kill it during next cell division)

Therefore Alkylating Agents can be used for the treatment of slow-growing tumors; extensive damage of DNA will lead to cell death

Nitrogen mustard first chemotherapeutic substance (interstrand-linking agent)

Nitrogen mustard drugs were developed from mustard gas,
a war agent first used during the First World War at Ypres. Could penetrate all protective materials except urethan One of the toxic-effects of exposure is the destruction of the bone marrow and white blood cells. Mustard gas Cure for cancer of the lymph glands Hodgkin's Disease.

Nitrogen mustard

Cisplatin
(Same strand-linking agent)

BETTER
than INTERstrand-linking agent!! because of two reasons

Cisplatin
(Same strand-linking agent)
1. Same strand cross-links formed are harder" to repair than cross-links between strands. 2. Cisplatin is able to replace zinc(II) ion in zinc-finger containing transcription factors, directly destroying transcription regulation Cisplatin Therapy Type of Cancer

Complete cure Sensitive

Responsive Resistant Activity shown

Testicular cancer
Ovarian

Bladder, Head & neck Cervix, Prostate, Esophegeal Various, eg.: Non-small cell lung, Ostegenic sarcoma, Hodgkins lymphoma

Synergy shown in combination with 5-fluorouracil, cytarabine and bleomycin, That allows for greater flexibility in the design of drug regimens.

Tumor could develop resistance to alkylating agents

Genomic changes in AA treated tumor cells may lead to: -- Increase in ability of treated cells to repair DNA defects; -- Decrease in cellular permeability to the Alkylating drugs;

-- Increase in glutathione synthesis conjugation with glutathione leads to chemical inactivation of alkylating agents (catalyzed by glutathione S-transferase)

II. Antimetabolite agents:

1. Antimetabolites are structural analogs of the naturally
occurring metabolites involved in DNA and RNA synthesis.

2. Antimetabolites exert their cytotoxic activity

either by competing with normal metabolites for the catalytic or regulatory site of a key enzyme or by direct substituting for a nucleotide that is normally incorporated into DNA and RNA.

3. Antimetabolites are most active in S phase cells

and have little effect on cells in G0. Consequently, these drugs are most effective in rapidly dividing tumors

Types of Antimetabolites:
Folate analogs -- Methotrexate (MTX)
(bind to catalytic site of dihydrofolate reductase DHFR)

Purine analogs -- Mercaptopurine,Fludarabine (inhibits enzymes involved in purine metabolism) Pyrimidine analogs -- Flurouracil (5-FU), Ara-C
(inhibits enzymes involved in pyrimidine metabolism)

Methotrexate (MTX)
www.vet.purdue.edu/ Looks very same to folic acid necessary nutrient.

Methotrexate
DHFR like MTX more than folic acid No production of No THF-co-enzymes tetrahydrofolic acid for one-carbon transfer No de novo purines and pyrimidines No DNA synthesis Treat leukemia, lymphomas, and osteosarcoma. It is also used in the treatment of AIDS and rheumatoid arthritis

Broad range

Methotrexate (MTX) resistance as an example of antimetabolite resistance

Genomic changes in MTX-treated tumor cells may lead to:

-- decrease in drug transport into the cell (less MTX)

-- alteration in gene DHFR encoding
dihydrofolate reductase enzyme with lower affinity for methotrexate (MTX-durable DHFR)

-- quantitative increase in DHFR concentration by DHFR gene amplification, or by increased DHFR mRNA production (more DHFR)

III. Plant derivates (heterogenous group)

Vinca alcaloids (Vinblastine, Vincristine) from periwinkle plant Vinca rosea (native to Madagaskar); Taxanes (Paclitaxel = Taxol and docetaxel = Taxotere) from needles of Pacific Yew plant Taxus brevifolia;

Podophyllotoxin (etoposide) from

Podophyllum peltatum (India) Camptothecins (topotecan, irinotecan) from Camptotheca acuminata (China)

Vinblastine, Vincristine
-- Mechanism of action = mitosis block: bind to tubulin and induce microtubule depolymerization (Disassembly)

Cells are arrested at metaphase as no chromosome segregation possible

http://www.img.cas.cz/dbc/gallery.htm
Red anti-tubulin; Green anti-vimentin; Blue -- DNA

Mouse 3T3 fibroblast (normal)

3T3 after vincristine

Vinca alcaloids bind to tubulin dimers at a specific recognition site on the protein. Tubulin form paracrystaline aggregates

Concentration of the free dimers is reduced

Mitotic spindles can not be formed

Equilibrium of growth/shrinkage of tubules shifted to the shrinkage

www.staff.ncl.ac.uk/i.r.hardcastle/ antibiotics.html

www.chem.wisc.edu/~bestchem/ taxol.gif

Paclitaxel (Taxol) and docetaxel (Taxotere)

-- Mechanism of action = mitosis block: promote microtubule assembly and super stability, action is opposite to vinca alcaloids, but effect is the same
Bundling of accumulated, disorganised microtubule filaments.

+ Taxol

Used for treatment of otherwise resistant ovarian cancers and recurrent breast tumors

Podophyllotoxin (etoposide) = Topo II inhibitor

Reconnection of DNA

Etoposide stabilizes TOPO II DNA complex in non-sealable form

Both ends of DNA can freely rotate within the enzyme

Etoposide inhibit topoisomerase II that catalyses transient breaking and re-joining of ds DNA strands (DNA becomes damaged

Camptothecins (topotecan, irinotecan) = Topo I inhibitor

topoisomerase I produces reversible single-strand breaks in DNA
These single-strand breaks relieve torsional strains, and allow DNA replication to proceed. Topotecan binds to the topoisomerase I-DNA complex and prevents ligation of the DNA strand, resulting in DNA breakage during the elongation and cell death TOPO I is not required for the viability of cells (TOPO II can substitute). Therefore, TOPO I negative cancer cells are viable and resistant to topotecan

IV. Antitumoral antibiotics (produced by fungi)

Anthracyclines: Doxorubicin (Adriamycin) and Daunorubicin Produced by fungus Streptomyces percetus var caesius.

Doxorubicin

Bleomycin Produced by Streptomyces verticillus as a mixture of small-molecular-weight copper-chelating glycopeptides

Bleomycin

Anthracyclines:
Doxorubicin (Adriamycin) and Daunorubicin

Mechanisms of action:
1) intercalation between DNA base pairs and inhibition of DNA topoisomerases I and II.

2) Altering membrane fluidity and ion transport

Active in Hodgkin's disease, non-Hodgkin's lymphomas, sarcomas, acute leukemia, and breast, lung, and ovarian carcinomas

Bleomycin
Mechanisms of action - multiple: 1) binds to double- and single-stranded DNA produces site-specific and non-specific SS and DS breaks ratio single:double = 10:1; Cleave DNA at G-C and G-T sequences; DNA in open chromatin esp. sensitive (where genes are expressed)

2) Makes non-covalent interstrand links

3) Inhibits DNA reparation by suppression of DNA ligase

Rapidly degraded by bleomycin hydrolase present in most tissues except skin and lung

No bone marrow and intestinal toxicity!! (Pulmonary fibrosis and skin effects are strong...)

Multidrug resistance phenomenon

Is a membrane associated phenomenon that represents a serious obstacle to chemotherapy of cancer Cell that are treated with (lets say) etoposide and develop resistance to etoposide, simultaneously become resistant to Methotrexate MDR caused by induction of membrane proteins that effectively and (almost) non-specifically expel small molecules from the cell! P-glycoprotein (170 kDa)
Belongs to traffic-ATPase superfamily of transport proteins

Other transporters BCRP, ABC 1,2,3 etc.

P-glycoprotein
This is an ATP-dependent export pump (= efflux pump) of broad specificity This efflux pump expel hydrophobic drugs, natural products and peptides.

The transport function of P-glycoprotein can be blocked by the action of another group of compounds known as MDR modulators, or chemosensitizers

cyclosporin A, verapamil, tamoxifen

RADICAL STRATEGIES of the TUMOR TREATMENT

Radiotherapy
Surgery Chemotherapy

IMMUNOTHERAPY

Hormonal therapy

RADIOTHERAPY
General principle is the same reduce a mass of rapidly dividing cells (cytoreduction principle, the same as for chemo)

Side effects are the same as for chemo

Difference radiotherapy is regional. IR could be focused like a beam of light to a treated area (internal or external)

HORMONAL THERAPY
Good for hormone-dependent tumors
Speaking generally, hormonal therapy can be discussed as trophic factor removal therapy
Estrogens are trophic factors for breast
epithelium and uterine epithelium

Removal of estrogen from ER-positive tumors leads to stop of their growth

Tamoxifen (Nolvadex)

Tamoxifen mimics the action of estrogene and binds to estrogen receptor (ER).

The tamoxifen-ER complex dimerises Than transported from the cytosol into the cell's nucleus The dimeric tamoxifen-ER complex binds to DNA

DNA-tamox-ER complex is unable to function in the same way as the DNA-estrogene-ER complex
In reality mechanism is not known completely !!!! In other tissues of the body Tamoxifen plays exactly like estrogene itself !!!

IMMUNOTHERAPY
Any intervention to enhance the body's natural ability to defend itself against malignant tumors. Cytokines: IFN-alpha, IL-2, Tumor necrosis factors (TNFs) Monoclonal antibodies: anti-CD20 Rituximab for lymphomas,
anti-HER2 trastuzumab (herceptin) for breast cancer

Cancer Vaccines: cancer-specific antigens +

other components boosting immunity (e.g. dendritic cell vaccines)

RADICAL TUMOR TREATMENT STRATEGIES

Radiotherapy
Surgery Chemotherapy

Hormonal therapy

IMMUNOTHERAPY

MODERN science-oriented therapies

Modern science for cancer treatment improvement

SURGERY PCR assisted surgery CHEMOTHERAPY new target-specific drugs, tumor-specific delivery
RADIOTHERAPY normal (p53 positive) cell can be protected by p53 inhibitor pifithrin

HORMONAL THERAPY designed chemical inhibitors of hormone receptors

IMMUNOTHERAPY entirely modern area