# Presentation: 467391

## TRAnslational research in Clinical Oncology (TRACO)

## Program Director

**Terry W. Moody, Ph.D.**
**(301)451-9451 **
**FAX(301)480-4323 **
**Bldg. 31, Rm. 4A48**

## Organizing Committee

**Irwin Arias**
**Pierre Henkart **
**Terry Moody**
**Jonathan Wiest**
**Lyuba Vartikovski**
**Course coordinator**
**Nauramy Osefo**

## SYLLABUS

**DATE****	****	****TOPIC****	****	****	****	****	****	****	****SPEAKERS****	**
**Sept. 8****	****Introduction, Lung Cancer****	****	****	****	****	****	****Moody, Giaccone**** **
**Sept. 15****	****Clinical Trials, Small Molecules****	****	****	****	****	****Janik, Austin****	**
**Sept. 22****	****HIV, Metastasis**
**	****	****	****	****	****Maldarelli, Hunter**
**Sept. 29****	****NO CLASS**
**Oct. 6****	****	****Angiogenesis, Ovarian Cancer**
**	****	****	****	****	****Zudaire, Annunziata**

**DATE****	****	****TOPIC****	****	****	****	****	****SPEAKERS****	****	**
**Oct. 13****	****NO CLASS**
**Oct. 20****	****Imaging, Prostate Cancer **** ****	****	****	****	****Choyke, Dahut**
**Oct. 27****	****Breast Cancer, Genomics**** ****	****	****	****	****Korde, Khan**
**Nov. 3****	****Lung cancer, Radiation Oncology****	****	****	****	****Szabo, Camphausen**** **
**Nov. 10****	****Immunology, Colon Cancer**
**	****	****	****	****	****Henkart, Viner**** **

**DATE****	****	****	****TOPIC****	****	****	****	****	****	****SPEAKERS****	**** **
**Nov. 17****	****	****Lymphomas, siRNA****	****	****	****	****	****Wilson, Caplen**
**Nov. 24****	****	****Inflammation, Epigenetics **** ****	**** ****	****	****	****	****	****Harris, Verma**
**Dec. 1****	****	****	****Health disparities, Epidemiology**** ****	**** ****	****	****	****	****Ambs, Caporaso**
**Dec. 8****	****	****Cervical cancer, Lung cancer vaccines **** ****	****	****	****	****	****Schiller, Morris**
**Dec. 15****	**** ****	****Cancer stem cells, Case reports **** ****	****	****	****	****	****Vonderhaar, TBA**** ****	****	****	**

**  ****The course is open to all interested personnel without charge.  Registration is available at the NCI CCR Web site**

## CCR component

**   ****Registrants can attend tumor** **boards, grand rounds, visit technology and/or core facilities.  Please contact Dr. Moody, if interested to make appropriate reservations.**

## COURSE CERTIFICATION

**   ****Registrants can obtain a course certificate upon passing a computer graded final examination.****	**

## Lung, colon, breast and prostate cancer account for half of the U.S. cancer mortalities.

**TYPE****	****	****INCIDENCE****	****(MORTALITY)**
**Lung****	****	****	****171,900****	****	****(157,200)**
**Colon/Rectum****	****147,500****	****	**** (57,100)**
**Breast****	****	****211,300****	****	**** (39,800)**
**Prostate****	****	****220,900****	****	**** (28,900)****	**
_**Others**__**	**__**	**__**582,500**__**	**__**	**__** (273,500)**_
**Total****	****	****	****1,334,100****	****	****(556,500)**
**Jemal, Ward and Thun, “Cancer: Principles & Practice of Oncology.”  Edited by DeVita, Hellman and Rosenberg. (2006), pp. 226-241**

## Cancers which kill 10,000-30,000 U.S. patients annually include:

**Pancreatic cancer**
**Non-Hodgkin’s Lymphoma**
**Leukemia**
**Stomach cancer**
**Ovarian cancer**
**Brain cancer**
**Liver cancer**
**Bladder cancer**
**Esophageal cancer**
**Kidney cancer**

## Cancer risks include:

**Alcohol**
**Asbestos**
**Diet**
**Familial**
**Hormones**

## Cancer risks (continued)

**Obesity**
**Ion Radiation**
**Tobacco**
**U.V. Radiation**
**Viral**

## Lung Cancer kills over 150,000 patients in the U.S. annually.

**There are 45 Million current smokers and 45 Million ex-smokers in the U.S.  **
**It is difficult to quit smoking due to nicotine addiction.**

## Carcinogens which have been identified in cigarette smoke include:

**Polyaeromatic hydrocarbons (PAH), **
**aza-arenes,**
**4(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK),**
**1,3 butadiene, **
**ethyl carbamate, **
**ethylene oxide, **
**nickel, chromium, cadmium, **
**polonium, arsenic**
**hydrazine**

## The process by which unreactive carcinogen converts to a form which binds DNA is known as metabolic activation.

**Bay region diol epoxides are the principal PAH metabolites involved in DNA adduct formation.  For Benz[a]pyrene (BaP), BaP-7,8-diol-9,10-epoxide (BPDE) forms adducts with DNA leading to G:C>T:A mutations in pulmonary DNA.  The genes for p53 and k-ras are frequently mutated.**

## BENZ(a)Pyrene

**BENZ(a)Pyrene**
**●****The chemical structure of BaP is shown.**

**	****BaP****               ****P450s**** ****Bap-7,8-oxide****               ****Epoxide hydrolase****BaP-7,8-diol****                      ****P450s****BPDE****              ****DNA****BPDE-N****2****-dG****               ****Acid hydrolysis****BP-tetraol****Boysen and Hecht, Mutation Res. 543:17(2003).**

**BaP is metabolized by enzymes to BPDE.**

## Carcinogens can be detoxified  and excreted prior to DNA damage.

**Cytochrome p450 enzymes catalyze addition **
**of an oxygen to the carcinogen, increasing **
**its water solubility.**
**●  ****Phase 2 enzymes convert the oxygenated **
**carcinogen to a form that is highly soluble in **
**water, converting it to a form that can be **
**excreted.**

## DNA is mutated if the rate of carcinogen activation exceeds the rate of carcinogen detoxification and/or DNA repair.

**DNA adducts as well as intra- and **
**interstrand DNA crosslinks are removed by nucleotide excision repair.**

## P53, a tumor suppressor gene:

- ●**mediates the G1 to S-phase checkpoint of the cell cycle,**
**●****drives programmed cell death or apoptosis after DNA damage,**
**●****is increased along with p21 after DNA damage.**

## P53 mutations are detected in most of the lung cancer patients.

**G to T transversions occur at the CpG rich codons including 153-158 (exon 5), 248 and 249 (exon7) and 273 (exon 8) of the p53 gene.  There is an excess of G to T transversions in smokers relative to non-smokers.**

## Slide 23

## Slide 24

## p53 mediates the G1 to S-phase checkpoint of the cell cycle

- ●DNA damage increases p21 and p53.
- ●P53 drives programmed cell death or apoptosis after DNA damage

## Slide 26

## Genotoxicity of tobacco smoke.

- ●**After 10 years of chronic cigarette smoking, normal lung tissue can undergo hyperplasia and metaplasia.**
**●****After 15 years, dysplasia can result.**
**●****After 20 years, a carcinoma in situ can form.**
**●****After 25 years, a malignant cancer can form.**

## Carcinogenesis●Cancer progression occurs over a period of decades.

## Normal lung●Carbon dioxide is exhaled from the lung whereas oxygen is inhaled.

## Hyperplasia●After exposure to tobacco smoke, hyperplasia can result.

## DysplasiaContinued exposure to tobacco smoke leads to dysplasia.

## Adenoma●Continued exposure to carcinogens leads to benign tumors such as adenomas.

## Adenocarcinoma●Chronic exposure to tobacco leads to malignant tumors such as adenocarcinoma.

## Tumor formation  ●Growth factors promote carcinogenesis. Progression factors lead to malignant tumors.

## Tumors●The primary cancer can undergo metastasis to distant organs.

**Carcinoma**
**Angiogenesis**
**Migration, Invasion and Metastasis.**

## Genetic abnormalities in lung cancer include:

**Mutation of tumor suppressor genes such as ****p53**
**Silencing of tumor suppressor genes such as ****p16, Rb****	****	****	****	**
**Amplification of oncogenes such as****	**
**	****c-myc, cyclin D1, EGF receptor, erbB-2****	****	****	**

## Loss of heterozygosity in lung cancer occurs on several genes.

**Gene locus****	****Tumor suppressor gene**
**	****3p12****	****	****	****	****FHIT**
**	****11q12****	****	****	****	****p15, p16**
**	****13q14****	****	****	****	****Rb**
**	****17q13****	****	****	****	****p53**

## Types of Lung Cancer.

**SCLC (small cell lung cancer)**
**NSCLC**
**	****adenocarcinoma**
**	****large cell carcinoma**
**	****squamous cell carcinoma**

## The epidermal growth factor receptor (EGFR) is overexpressed in NSCLC.

**Cell Type****	****	****	****Density (fmol/10****6 ****cells)**
**Adenocarcinoma****	****	****	****8.4**
**Large cell Carcinoma****	****	****	****7.0**
**Squamous cell Carcinoma****	****	****6.2**
**SCLC****	****	****	****	****	****<0.2**
**125****I-EGF bound specifically to NSCLC cells (Moody et al.,J. Cell Biochem 43:445 (1990).**

## Tyrosine kinase receptors●Tyrosine kinase receptors can have Cys rich domains, multiple subunits, or Ig domains.  Biology of the Cell, Alberts et al., 2001.

## The EGFR is an 1186 amino acid integral membrane protein.

**The 621 amino acid extracellular domain binds EGF with high affinity.**
**The 24 amino acid transmembrane domain anchors the receptor into the membrane and tranduces signaling.**
**The 541 amino acid intracellular domain contains tyrosine kinase activity.**
**Lys721 binds ATP and Tyr amino acids are subsequently phosphorylated.**
**Tyr1068, 1086, 1148, 1174 are autophosphorylated**

## Tyrosine kinase receptors cause increased cell survival.●Tyrosine kinase receptors activate Akt. Molecular biology of the cell: Alberts et al., 2001.

## EGF, TGFα and mAb 108 bind with high affinity to lung cancer cells.

**Agent****	****	****	****IC****50****, ug/ml**
**EGF****	****	****	****	****.03**
**TGF********	****	****	****.8**
**TGF********-PE38****	****	****.4**
**mAb 108****	****	****	****3**
**IgG****	****	****	****	****>10**
**The IC****50**** to inhibit ****125****I-EGF specific binding to NCI-H157 cells was determined.**

## EGFR tyrosine phosphorylation ●Addition of EGF to lung cancer cells causes EGFR, PLC, and PI-3-K tyrosine phosphorylation.

## The EGFR regulates cancer cell growth.  ●The EGFR activates ERK leading to increased proliferation of cancer cells.  Molecular Biology of the cell; Alberts et al., 2001.

## EGF stimulates and mAb 108 inhibits colony formation.

**Addition****	****	****	****Colony number**
**None****	****	****	****	****66 **_**+**_** 4l**
**EGF, 0.1 ug/ml****	****	****156 **_**+**_** 24***
**Mab 108, 1 ug/ml****	****	****32 **_**+**_** 1***
**EGF + Mab 108****	****	****71 **_**+**_** 5**
**IgG, 1 ug/ml****	****	****	****85 **_**+**_** 12**
**The mean value **_**+**_** S.D. of 3 determinations is indicated, p < 0.05**

## Slide 47

## MAb-108 localizes to lung cancer xenografts.

**Organ****	****	****	****cpm/mg wet tissue**
**Tumor****	****	****	****4.6 **_**+**_** 0.5**
**Lung****	****	****	****	****2.8 **_**+**_** 0.1**
**Stomach****	****	****	****2.4 **_**+**_** 0.1**
**Spleen****	****	****	****2.2 **_**+**_** 0.2**
**Heart****	****	****	****	****2.1 **_**+**_** 0.5**
**Liver****	****	****	****	****1.8 **_**+**_** 0.1**
**Intestine****	****	****	****0.9 **_**+**_** 0.1**
**Brain****	****	****	****	****0.1 **_**+**_** 0.1**
**The mean value **_**+**_** S.D. of 3 determinations is indicated**

## EGFR mutations occur in Japanese lung cancer patients.

**In-frame deletions of EGFR amino acids **
**718-729 result in increased patient **
**response to gefitinib, a tyrosine kinase **
**inhibitor.  **
**Lynch et al., NEJM 350:2129 (2004)**

## U.S. patients which have few EGFR mutations are less responsive to Gefitinib or Iressa.

**EGFR mutations occur in the activation loop, especially L858R and G719S.**
**   ****Paez et al., Science 304:1497 (2004)**

## The EGFR dimerizes prior to internalization.

**Deletion mutations in exon 19 may be **
**recessive and homodimers may be needed **
**for excessive tyrosine phosphorylation.**
** ●****Point mutations in exons 18 and 21 may **
**be dominant and heterodimers may have **
**excessive activity.**
**Minna et al., Science 304:1458 (2004).**

## Tyrosine kinase receptors are molecular targets for the development of small molecules.

**● ****GleevecTM (STI571), which blocks bcr-abl tyrosine kinase activity, is being used to treat chronic myelogenous leukemia (CML).**

## CML was treated with interferon-α.

**This restores blood counts in **
**approximately 70% of the patients and **
**delays disease progression in 20%**
**of the patients.****	**

## CML patients have a genetic abnormality on chromosome 22 (Philadelphia chromosome). ●Segments of chromosome 9 and 22 are fused resulting in the bcr-abl gene. ●The resulting tyrosine kinase is constituitively active.  ●Bcr-abl tyrosine kinase actvity is inhibited by GleevecTM.

## Translocation of Bcr/Abl.  ●Chromosome 22 translocates with chromosome 9.  Molecular Biology of the Cell; Alberts et al., 2001.

## Translocation of Bcr-Abl Genes

**Translocation of *****Bcr-Abl***** Genes**
**●****Translocated chromosome 9 appears larger and translocated chromosome 22 appears smaller: Freebies for Teachers”; D. Kerrigan.**

**Fusion protein with tyrosine kinase activity**

**(q+)**

**Ph**

**(22q–)**

***bcr-abl***

***abl***

***bcr***

**22**

**9**

**9**

**Notes:**
In chronic myelogenous leukemia, a translocation occurs between chromosomes 9 and 22. This rearrangement of genomic material creates a fusion gene call Bcr-Abl that produces a protein (tyrosine kinase) thought to promote the development of leukemia. The drug Gleevec blocks the activation of the Bcr-Abl protein.

## In a Phase I Clinical Trial,GleevecTM was effective orally at a daily dose of 300 mg or greater.

**Dose limiting toxicities included nausea, vomiting, edema and rash. ****(Sawyers and Druker. Cancer J. Sci. Am. 1999;5:63).**

## In a Phase II Clinical Trial,GleevecTM restored normal blood counts in 53 out of 54 chemotherapy-resistant CML patients.

**After a year on Gleevec, 51 of **
**these patients were still doing well. ****(Druker et al. N. Engl. J. Med. 2001; 344: 1038.).**

## In 2002, the Food and Drug Administration (FDA) approved  Trial, (Imatinib mesylate) for a rare stomach cancer called gastrointestinal stromal tumor (GIST). ●GleevecTM inhibits tyrosine kinase activity of c-KIT in GIST.  ●Some shrinkage of the tumors was seen in 59% of the patients while another 26% had tumors that stopped growing.  ●Side effects included swelling of the legs and eyes and tumor bleeding.

## Gleevec mechanism of action  ●Gleevec blocks the ATP binding site.  Molecular biology of the cell; Alberts et al., 2001.

**PRACTICAL STEPS TO PREVENT CANCER**
**●****Check your house for radon.**
**●****Check your house for asbestos.**
**●****Take precautions at your workplace.**
**●****Check your community water system.**
**●****Avoid breathing polluted air.**
**●****Protect your skin.**
**●****Don’t breathe smoke.**
**●****Exercise daily.**

**PRACTICAL STEPS TO PREVENT CANCER (continued)**
**●****Avoid pesticides.**
**●****Eat fruits and vegetables.**
**●****Reduce red-meat consumption.**
**●****Eat fish.**
**●****Minimize fried foods.**
**●****Drink alcohol in moderation.**
**●****Avoid unnecessary x-rays.**
**●****Reduce infections.**