1. Genetic Alterations in Cancer Original slides by: Dr. B. F. Burns Dr. Bojana Djordjevic Department of Pathology and Laboratory Medicine

2. Objectives Discuss the common types of cancer- associated genes and provide examples of each, discussing their normal function and their effects when normal function is lost. Describe the common types of changes that can affect genes associated with cancer.

3. Genetic Alterations in Cancer All neoplasms result from genetic changes in the tumor cells Changes may be: inherited - “cancer kindreds” (relatively rare) mutations are in the “germ line” acquired (most common) mutations are only in the tumor cells

4. Features common to cancer cells 1.Growth in the absence of “go” signals 2.Growth despite “stop” signals issued from neighboring cells 3.Evasion of “auto-destruct” pathways in response to genetic damage 4.Stimulate local blood vessel growth 5.Effectively immortal 6.Locally invasive growth and metastases

5. Hanahan and Weinberg, Cell, 2000.

6. Basic Concepts in Oncogenesis Monoclonality initial mutation occurs in a single cell mutated cell is effectively “immortalized”, either by replicating uncontrollably or not dying off normally replication of this cell results in “(mono)clonal expansion”

7. Basic Concepts in Oncogenesis Tumour progression initial mutation is NOT sufficient to produce a clinical “tumour” genome appears to be unstable (? DNA repair defect) leading to further mutations sequential mutations lead to subclones with progressively more “malignant” phenotypes (a nasty form of natural selection)

8. Hanahan and Weinberg, Cell, 2000.

9. Tumor cells begin as a clone, but become progressively more heterogeneous with time T T T N T T T T+ 1. Initial mutation starts immortalized clone - not fully malignant yet 2. Second, third, etc. mutations give further growth advantages to sub-clones T+ T T T* T T+ T* T T T T T+ T* 3. Mature tumor contains many different sub- clones After Robbin’s Pathologic Basis of Disease

10. Tumour cell kinetics Tumour cells don’t always divide more quickly than normal cells “Growth fraction” (proportion of cells actively dividing) usually increased tumour cells don’t die normally (growth by accumulation)

11. Classes of Genes involved in Cancer Cell cycle genes – control cell replication Growth factors and receptors Signal transduction proteins Nuclear regulatory proteins Tumor suppressor genes Apoptosis (programmed cell death) proteins and normal senescence (telomeres) DNA repair genes

12. Cancer Associated Genes All of these genes exist and function in normal cells as 1. proto-oncogenes 2. tumor suppressor genes 3. apoptosis genes. depending on their normal function they may contribute to a cancer by overactivity or underactivity and may behave in a dominant or recessive fashion in the cell. Overactivity may result from increased expression or decreased degradation (e.g.. mutations that prolong protein half-life)

13. Types of Genes associated with cancer “Classic” Oncogenes promote cell growth and division ras, myc and Her2/neu are examples Tumour Suppressor Genes normally inhibit cell division p53, Rb and BRCA-1 and -2 are examples Apoptosis Genes associated with normal cell death and turnover bcl-2 family is best known

14. “Classic” Oncogenes Mutation or overexpression associated with overactivity of gene genetically dominant effect example: ras point mutations are very common in human tumours mutant ras signal transducer doesn’t need growth factor binding to be active

15. Sites of action of “classic” oncogenes Growth factors Growth factor receptors Signal transduction proteins Nuclear regulatory proteins Cell cycle regulators

16. Mechanisms of Oncogene action - Growth Factor overexpression Saturated receptors send excess growth signals Overexpression of sis

17. Mechanisms of Oncogene action - Growth Factor Receptor overproduction Her2/neu overexpressed Excess receptors make cells overly sensitive to existing growth factors

18. Her2 +

19. Mechanisms of Oncogene action – Signal Transducer Mutations Mutant ras doesn’t need external activation Signals activate transcription and cell division Receptor on surface bypassed

20. Mechanisms of Oncogene Action - Mutated Nuclear Transcriptional Activators Myc protein Surface receptors bypassed Myc protein 1. Mutated Transcriptional Activator gene 2. Excess myc production 3. Deregulated cell replication

21. Mechanisms of oncogene action - Cell cycle proteins Cyclin D and the Cyclin-dependent kinases G1G1 G2G2 S M cdk4 Cyclin D Active complex controls G 1 to S via Rb phosphorylation Overexpression of Cyclin D or cdk4 leads to loss of normal control of cell replication, occurs in many different tumors

22. Tumor Suppressor Genes - General features genes that normally function to suppress cell replication (not specifically to prevent tumors) can be considered to function in opposition to effects of “classic” oncogenes normal cell growth depends on a balance between the effects of proto-oncogenes and TSG’s

23. Tumour Suppressor Genes - Genetics Only need one good copy (wild type) of the gene to maintain function “recessive” genetics at a cellular level Theory may have to be modified slightly to recognize “haplo-insufficiency” With one allele being mutated, a chance mutation of the second normal allele results in both functioning alleles being knocked out - referred to as “loss of heterozygosity”. The function of the TSG is lost and tumor develops. TSG defects inherited in a dominant fashion ALL the family members who carry one mutated allele are at risk of cancer

24. Tumour Suppressor Genes - Genetics Tumors involving TSG Familial Carry one mutated allele in the germline Tumor arises when there is a sporadic mutation of the other allele (loss of heterozygosity) Early age of onset Multifocal Bilateral Sporadic Both wild type alleles must undergo sporadic mutations in order for tumor to develop Later in life Unifocal Unilateral

25. Retinoblastoma (Rb): The “original” tumour suppressor gene First recognized TSG (1968), familial inheritance of infantile eye tumours Knudson proposed that both alleles of the gene had to be mutated in order to see the effect (“two hit hypothesis”)

26. Clinical appearance of retinoblastoma - leukocoria Normal “red eye” Leukocoria – white eye

27. Familial inheritance of Retinoblastoma Heterozygous Zygote: 1 in 2 chance Normal gene Mutant Rb gene Chance 2’nd mutation- virtual certainty Homozygous mutant cell Millions of cell divisions Retinoblastoma forms from that cell After Robbin’s Pathologic Basis of Disease

28. P53 - the prototype tumour suppressor gene p53 is the most common gene affected in human cancer normally functions as the “Guardian of the Genome” mutagen exposure leads to increased T 1/2 of p53, cell is blocked in G 1, allowing for DNA repair if repair fails, p53 induces apoptosis genes, cell dies

29. Genes affecting Apoptosis another way in which tumours may form is if a clone of cells fails to die in a normal fashion (apoptosis) the genes controlling this may either prevent apoptosis (e.g.. bcl-2 ) or induce it (e.g.. bax) within a cell the balance of these two determines whether the cell goes into apoptosis

30. How Genes affecting Apoptosis act to produce tumors bcl-2 protein functions to block programmed cell death (apoptosis) abnormal expression “immortalizes” the cell such cells are at increased risk for additional mutations of other oncogenes t(14;18) translocation in follicular lymphomas leads to increased bcl-2 expression

31. DNA-repair defects Not actually oncogenic proteins, but inability to repair ongoing mutations to other proto-oncogenes predisposes to cancer Initially discovered via familial syndrome of ataxia-telangiecatasia - ATM protein senses DNA damage, activating p53 cascade Mismatch repair protein defects in hereditary non-polyposis colon cancer Xeroderma pigmentosa patients cannot repair UV-light DNA damage

32. The Colon Cancer Sequence

33. Multistep mutations in Adenoma to Colon Cancer – “Suppressor Pathway” Normal Epithelium APC gene lost on 5q Hyperplastic Epithelium Ras mutation on 12 Low grade adenoma Intermediate grade adenoma DCC loss on 18 High grade adenoma p53 loss on 17 Carcinoma Metastases ? others After Volgelstein & Fearon et al NEJM 1988;319:525-532 DNA methylation loss

34. Pathologic Correlates in the Colon Cancer Sequence Benign Tubular Adenoma of Colon Colonic Carcinoma arising in a pre-existing Adenoma Adenoma Carcinoma going through wall of colon Stalk of normal colon mucosa

35. Microscopic correlates in the Adenoma - Cancer sequence High grade Dysplasia of Epithelium Normal Epithelium Low grade Dysplasia Adenoma Carcinoma

36. Second mechanism of colon cancer development “Mutator pathway” – involved in 10-15% of all colon cancers and in all of the sub- group called “Hereditary non-polyposis colon cancer” Involves mutations to mismatch repair genes leading to “microsatellite instability” (MSI) in the genome Clinical importance is in: pointing to family genetic testing for MSI typically lower stage/better prognosis Jass, Whitehall, et al. Gastroenterology 2002; 123: 862-876

37. Types of Genetic Changes affecting Cancer-associated genes Point Mutations- activating proto-oncogenes e.g. ras Inactivating tumor-suppressor genes, e.g. RB Aneuploidy - encompasses gross chromosomal changes with truncations, extensions or swapped segments such as: Chromosomal Translocations- resulting in increased expression (bcl-2) or abnormal “fusion” protein (bcr-abl) Gene Amplifications- increased copy number of genes, e.g. n-myc and Her2/neu Chromosomal deletions- esp. affecting tumor suppressor genes, e.g. Rb deletions at 13q14

38. Chromosomal Translocations Most common in leukemias and lymphomas Many are characteristic of specific types: t(9;22) translocation in chronic myelogenous leukemia t(8;14) in Burkitt lymphoma t(14;18) in follicular lymphoma

39. Chromosomal Translocations From Robbin’s Pathologic Basis of Disease, 5’th Ed.

40. Chromosomal Translocations From Robbin’s Pathologic Basis of Disease, 5’th Ed.

41. Gene Amplification An increase in the copy number of certain oncogenes leads to over-expression Her2/neu most common example n-myc amplification is common in pediatric neuroblastomas larger copy number is associated with worsening prognosis

42. Summary points: Genetic changes are at the root of all cancer A given type of cancer may arise and evolve genetically in an extremely variable fashion Within a single tumor the genetic abnormalities are variable from cell to cell

43. Further reading Robbins and Cotran Pathologic Basis of Disease. 9’th Ed. 2015. Pages 282-306. This is the portion of the Neoplasia chapter dealing with genetic alterations and cancer-associated genes.