Carcinogenesis

CARCINOGENESIS
By- Dr. Chirag Patil
[email protected]
M.D.S in Oral & Maxillofacial Surgery

1. CARCINOGENESIS B y – Dr. Chirag Patil M.D.S in Oral and Maxillofacial Surgery

2. Carcinogenesis or oncogenesis or tumorigenesis means mechanism of induction of tumours (pathogenesis of cancer); agents which can induce tumours are called carcinogens A mass of tissue formed as a result of abnormal,excessive,uncordinated , autonomous and purposeless proliferation of cell even after cessation of stimulus for growth which caused it is called Neoplasia

3. Etiology and pathogenesis of cancer is discussed under the following 4 broad headings: A. Molecular pathogenesis of cancer (genes and cancer) B. Chemical carcinogens and chemical carcinogenesis C. Physical carcinogens and radiation carcinogenesis D. Biologic carcinogens and viral oncogenesis .

4. A. MOLECULAR PATHOGENESIS OF CANCER (GENETIC MECHANISMS OF CANCER) Basic Concept of Molecular Pathogenesis: How a normal cell is transformed to a cancer cell-

5. 1. Monoclonality of tumours - most human cancers arise from a single clone of cells by genetic transformation or mutation. 2. Field theory of cancer- In an organ developing cancer, in the normal cells, limited number of cells only grow into cancer after undergoing sequence of changes under the influence of etiologic agents . 3. Multi-step process of cancer growth and progression Gradual multi-step process involving many generations of cells. The various causes act on the cell one after another ( multi-hit process). Further progression of the tumour . Ultimately, the cells transformed having features of malignancy—excessive growth, invasiveness and distant metastasis

6. 4. Genetic theory of cancer. Cell growth of normal as well as abnormal types is under genetic control. In cancer, there are either genetic abnormalities in the cell , or there are normal genes with abnormal expression. The abnormalities may be from inherited or induced mutations (carcinogenic agents: chemicals, viruses, radiation). The mutated cells transmit their characters to the next cells and result in cancer.

7. 5. Genetic regulators of normal and abnormal mitosis. Regulatory genes control mitosis & cell aging, terminating in cell death by apoptosis. 4 regulatory genes: Oncogenes - Mutated form of normal protooncogenes in cancer i ) Proto- oncogenes are growth-promoting genes ii ) Anti- oncogenes are growth-inhibiting or growth suppressor genes. iii) Apoptosis regulatory genes control the programmed cell death. iv) DNA repair genes, that has occurred during mitosis and also control the damage to proto- oncogenes and antioncogenes .

8. In cancer, - Abnormal cell growth due to genetic damage Abnormalities in 4 cell regulatory genes are as under: Activation of growth-promoting oncogenes Inactivation of cancer-suppressor genes Abnormal apoptosis regulatory genes Failure of DNA repair genes

9. Cancer-related Genes and Cell Growth (Hallmarks of Cancer)

10. 1. EXCESSIVE AND AUTONOMOUS GROWTH: GROWTH PROMOTING ONCOGENES protooncogenes become activated oncogenes by following mechanisms : Mutation in the protooncogene which alters its structure and function. Retroviral insertion in the host cell. Damage to the DNA sequence that normally regulates growth-promoting signals of protooncogenes resulting in its abnormal activation . By formation of extra copies of protooncogene causing overproduction that promotes excessive cellular proliferation .

11. Transformation of proto-oncogene to oncogenes occur by three mechanisms: i ) Point mutations- alteration of a single base in the DNA chain ii) Chromosomal translocations - transfer of a portion of one chromosome carrying proto-oncogene to another chromosome and making it independent of growth controls. iii) Gene amplification - increasing the number of copies of DNA sequence in proto-oncogene leading to increased mDNA and thus increased or over expressed gene product.

12. Growth factors (GFs). GFs were the first proto- oncogenes to be discovered for cell proliferation cascade. Bind to cell surface receptors to activate cell proliferation cascade within the cell. However, a cancer cell may synthesise a GF and respond to it as well, cancer cells acquire growth self-sufficiency. Growth factor genes are not altered or mutated . large secretion of GFs which stimulate cell proliferation.

13. Mechanisms of activation of proto- oncogenes to form growth promoting oncogenes .

14. Important Oncogenes , their Mechanism of Activation and Associated Human Tumours

15. REFRACTORINESS TO GROWTH INHIBITION: GROWTH SUPPRESSING ANTI-ONCOGENES Mutated anti- oncogenes behave like growth-promoting oncogenes Important Tumour -suppressor Anti- oncogenes Retinoblastoma protein (RB Gene) P53 gene Transforming growth factor- ß (TGF- ß) and its receptors Adenomatous polyposis coli(APC) gene & ß- catein protein Von- hippel - lindau (VHL) Willms tumour (WT gene) Neurofibroma gene

16. Oncogenes versus Antioncogenes .

17. ESCAPING CELL DEATH BY APOPTOSIS: GENES REGULATING APOPTOSIS AND CANCER Another mechanism of tumour growth is escaping cell death by apoptosis . Apoptosis in normal cell is by cell death receptor ( CD95) , resulting in DNA damage. other pro-apoptotic factors (BAD, BAX, BID and p53) apoptosis-inhibitors (B-Cell Lymphoma 2, BCL-X). In cancer cells, the function of apoptosis is interfered due to mutations in BCL2 & CD95

18. AVOIDING CELLULAR AGEING: TELOMERES AND TELOMERASE IN CANCER After each mitosis (cell doubling) there is progressive shortening of telomeres which are the terminal tips of chromosomes. Telomerase is the RNA enzyme helps in repair of such damage to DNA and maintains normal telomere length in successive cell divisions. after repetitive mitosis for a maximum of 60 to 70 times, telomeres are lost in normal cells and the cells cease to undergo mitosis. Cancer cells , telomere length is maintained. cancer cells avoid aging,mitosis does not slow down or cease

19. CONTINUED PERFUSION OF CANCER: TUMOUR ANGIOGENESIS Cancers can only survive if the cancer cells are adequately nourished and perfused , as otherwise they cannot grow further. Neovascularisation in the cancers not only supplies the tumour with oxygen and nutrients , but the newly formed endothelial cells also elaborate a few growth factors for progression of primary as well as metastatic cancer.

20. CONTINUED PERFUSION OF CANCER: TUMOUR ANGIOGENESIS The stimulus for angiogenesis is provided by the release of various factors: i ) Promoters of tumour angiogenesis- - vascular endothelial growth factor (VEGF) - basic fibroblast growth factor ( bFGF ). ii) Anti-angiogenesis factors- thrombospondin-1, - angiostatin , - endostatin - vasculostatin Mutated form of p53 gene in various cancers results in removal of anti- angiogenic role thus favouring continued angiogenesis

21. INVASION AND DISTANT METASTASIS: CANCER DISSEMINATION

22. DNA DAMAGE AND REPAIR SYSTEM: MUTATOR GENES AND CANCER Normal cells during complex mitosis suffer from minor damage to the DNA which is detected and repaired before mitosis is completed so that integrity of the genome is maintained. small mutational damage to the cell by exogenous factors (e.g. by radiation,chemical carcinogens etc) is also repaired. p53 gene is responsible for detection and repair of DNA damage. If system of DNA repair is defective as happens in some inherited mutations ( mutator genes), the defect in unrepaired DNA is passed to the next progeny of cells and cancer results.

23. CANCER PROGRESSION AND HETEROGENEITY: CLONAL AGGRESSIVENESS Biology of cancers is that with passage of time cancers become more aggressive;( tumour progression). Clinical parameters of cancer progression are: - increasing size of the tumour , higher histologic grade - areas of tumour necrosis - invasiveness and distant metastasis.

24. CANCER PROGRESSION AND HETEROGENEITY: CLONAL AGGRESSIVENESS molecular biology, passage of time cancer cells acquire more and more heterogeneity. cancer cells remain monoclonal in origin, they acquire more and more mutations which, in turn, produce multiple-mutated subpopulations of more aggressive clones of cancer cells (i.e. heterogeneous cells) in the growth which have tendency to invade, metastasise

25. CANCER—A SEQUENTIAL MULTISTEP MOLECULAR PHENOMENON: MULTISTEP THEORY cancer occurs several sequential steps of abnormalities in the target cell- Initiation promotion Progression multiple steps are involved at genetic level by which cell proliferation of cancer cells is activated- activation of growth promoters loss of growth suppressors inactivation of intrinsic apoptotic mechanisms escaping cellular aging

26. MICRORNAs IN CANCER: ONCOMIRS MicroRNAs ( miRNAs ) are evolutionally conserved, endogenous, noncoding single stranded RNA molecules with a length of 22 nucleotides only. Normally, miRNAs function as the regulators of cell proliferation, differentiation and survival. miRNAs have an oncogenic role in initiation and progression of cancer and are termed as oncogenic microRNAs , abbreviated as oncomiRs . oncomiRs can perform various functions : - tumour suppressor - tumour promoter -pro-apoptotic.

27. CHEMICAL CARCINOGENESIS

28. Stages in Chemical Carcinogenesis 1. INITIATION OF CARCINOGENESIS 2. PROMOTION OF CARCINOGENESIS 3. PROGRESSION OF CARCINOGENESIS

29. Stages in Chemical Carcinogenesis INITIATION OF CARCINOGENESIS first stage in carcinogenesis induced by initiator chemical carcinogens. The change can be produced by a single dose of the initiating agent for a short time, though larger dose for longer duration is more effective. The change so induced is sudden, irreversible and permanent.

30. Chemical carcinogens acting as initiators of carcinogenesis can be grouped into 2 categories. Direct-acting carcinogens Indirect - acting carcinogens

31. Direct-acting carcinogens. These are a few chemical substances (e.g. alkylating agents, acylating agents) which can induce cellular transformation without undergoing any prior metabolic activation a) Alkylating agents Anti-cancer drugs (e.g. cyclophosphamide, chlorambucil, busulfan , melphalan, nitrosourea etc ) β- propiolactone -Lymphomas Epoxides -AML b) Acylating agents –(Bladder cancer) Acetyl imidazole Dimethyl carbamyl chloride

32. Indirect-acting carcinogens or procarcinogens These require metabolic conversion within the body so as to become ‘ultimate’ carcinogens having carcinogenicity e.g . -polycyclic aromatic hydrocarbons, - aromatic amines, - azo dyes, - naturally-occurring products and others. In either case, the following steps are involved in transforming ‘the target cell’ into ‘the initiated cell’:

33. INDIRECT-ACTING CARCINOGENS (PROCARCINOGENS) a ) Polycyclic, aromatic hydrocarbons (in tobacco, smoke, fossil fuel, soot, tar, minerals oil, smoked animal foods, industrial -Lung cancer and atmospheric pollutants) -Skin cancer • Anthracenes ( benza -, dibenza -, dimethyl benza -) -Cancer of upper aerodigestive tract • Benzapyrene • Methylcholanthrene b) Aromatic amines and azo-dyes • β - naphthylamine -Bladder cancer • Benzidine • Azo-dyes (e.g. butter yellow, scarlet red etc ) -Hepatocellular carcinoma

34. c) Naturally-occurring products • Aflatoxin B 1 • Actinomycin D • Mitomycin C -Hepatocellular carcinoma • Safrole • Betel nuts d) Miscellaneous • Nitrosamines and nitrosamides - Gastric carcinoma • Vinyl chloride monomer -Angiosarcoma of liver • Asbestos - Bronchogenic carcinoma, mesothelioma • Arsenical compounds - Cancer, skin, lung • Metals (e.g. nickel, lead, cobalt, chromium etc ) - Lung cancer • Insecticides, fungicides (e.g. aldrin, dieldrin, chlordane etc ) - Cancer in experimental animals • Saccharin and cyclomate s

35. Sequential stages in chemical carcinogenesis ( left) in evolution of cancer (right).

36. 2. PROMOTION OF CARCINOGENESIS Promotion is the next sequential stage in the chemical carcinogenesis. Promoters of carcinogenesis are substances such as phorbol esters, phenols, hormones, artificial sweeteners and drugs like phenobarbital . They differ from initiators in the following respects : i ) They do not produce sudden change. ii) They require application or administration, for sufficient time and in sufficient dose. iii) The change induced may be reversible. iv) They do not damage the DNA per se and are thus not mutagenic but instead enhance the effect of direct-acting carcinogens or procarcinogens. v) Tumour promoters

37. 3. PROGRESSION OF CARCINOGENESIS Progression of cancer is the stage when mutated proliferated cell shows features of malignancy. These features pertain to morphology, biochemical composition and molecular features of malignancy. features appear only when the initiated cell starts to proliferate rapidly and in the process acquires more and more mutations. The new cells develops after such repetitive proliferation inherits genetic and biochemical characteristics of malignancy.

38. B. PHYSICAL CARCINOGENESIS Divided into 2 groups: Radiation- ultraviolet light and ionising radiation - carcinogenic agent. 2 . Non-radiation- Non-radiation physical agents are the various forms of injury

39. The main source of UV radiation- - sunlight -UV lamps -welder’s arcs. UV light penetrates the skin for a few millimetres that its effect is limited to epidermis. can cause various forms of skin cancers— - squamous cell carcinoma - basal cell carcinoma -malignant melanoma.

40. MECHANISM-

41. ULTRAVIOLET LIGHT. Xeroderma pigmentosum is predisposed to skin cancers at younger age (under 20 years of age). b) Ataxia telangiectasia is predisposed to leukaemia . c) Bloom’s syndrome is predisposed to all types of cancers. d) Fanconi’s anaemia with increased risk to develop cancer.

42. IONISING RADIATION. Ionising radiation - X-rays, - Alpha, - beta - Gyama -rays, - Radioactive isotopes - Protons and neutrons can cause cancer in man. Radiation-induced cancers- - leukaemias (except chronic lymphocytic leukaemia ); - thyroid (most commonly papillary carcinoma), -skin, breast, ovary, uterus, lung, myeloma, and salivary glands The risk is increased by higher dose and with high LET (linear energy transfer) such as in neutrons and Alpha-rays than with low LET as in X-rays and gyama -rays

43. Mechanism. Radiation damages the DNA of the cell by one of the 2 possible mechanisms : a ) It may directly alter the cellular DNA. b) It may dislodge ions from water and other molecules of the cell and result in formation of highly reactive free radicals that may bring about the damage . Damage DNA resulting in mutagenesis May cause chromosomal breakage, translocation, or point mutation. Effect depends upon a number of factors such as type of radiation, dose, dose-rate, frequency and various host factors such as age, individual susceptibility, immune competence, hormonal influences and type of cells irradiated .

44. BIOLOGIC CARCINOGENESIS Parasites. Schistosoma haematobium infection of the urinary bladder - squamous cell carcinoma of the urinary bladder Clonorchis sinensis , lives in the hepatic duct cause cholangiocarcinoma . Fungus. Aspergillus flavus - hepatocellular carcinoma Bacteria. Helicobacter pylori - chronic gastritis and peptic ulcer; its prolonged infection may lead to gastric lymphoma and gastric carcinoma.

45. Viral Carcinogenesis 20% of all cancers worldwide are due to persistent virus infection. oncogenic viruses with neoplasia was first observed by an Italian physician Sanarelli in 1889. The contagious nature of the common human wart was first established in 1907. .

46. Oncogenic Viral Infections: General Aspects viral infections (including oncogenic viruses) can be transmitted by one of the 3 routes: i ) Horizontal transmission- viral infection passes from one to another- - Direct contact - Ingestion of contaminated water or food - Inhalation. Most of these infections begin on the epithelial surfaces, spread into deeper tissues, and then through haematogenous or lymphatic or neural route disseminate to other sites in the body.

47. ii) By parenteral route - by inter-human spread and from animals and insects to humans. iii) Vertical transmission- when the infection is genetically transmitted from infected parents to offsprings . oncogenic viruses fall into 2 broad groups: -DNA oncogenic virus -RNA oncogenic viruse or retroviruses.

48. Both types of oncogenic viruses usually have 3 genes and are abbreviated according to the coding pattern by each gene: i ) gag gene : codes for group antigen. ii) pol gene : codes for polymerase enzyme. iii) env gene : codes for envelope protein .

49. Primary viral infections - majority of the common viral infections lasts for a few days to a few weeks and produce clinical manifestations. generally cleared by body’s innate immunity and specific immune responses. immunocompetent host is generally immune to the disease or reinfection by the same virus. However, body’s immune system is not effective against surface colonization or deep infection or persistence of viral infection. Persistence of viral infection or latent infection some viruses may occur by acquiring mutations in viruses which resist immune attack by the host, or induces immunosuppression in the host such as HIV.

50. oncogenesis by each group of DNA and RNA oncogenic viruses is briefly considered below: 1.Mode of DNA viral oncogenesis . 2. Mode of RNA viral oncogenesis .

51. 1.Mode of DNA viral oncogenesis . A, Replication: Step 1. The DNA virus invades the host cell . Step 2 . Viral DNA is incorporated into the host nucleus and T-antigen is expressed immediately after infection. Step 3. Replication of viral DNA occurs and other components of virion are formed. The new virions are assembled in the cell nucleus. Step 4. The new virions are released, accompanied by host cell lysis . B, Integration : Steps 1 and 2 are similar as in replication. Step 3 . Integration of viral genome into the host cell genome occurs which requires essential presence of functional T-antigen. Step 4 . A ‘ transformed ( neoplastic ) cell’ is formed.

52. 2. Mode of RNA viral oncogenesis . Step 1 . The RNA virus invades the host cell. The viral envelope fuses with the plasma membrane of the host cell; viral RNA genome as well as reverse transcriptase are released into the cytosol . Step 2 . Reverse transcriptase acts as template to synthesise single strand of matching viral DNA which is then copied to form complementary DNA resulting in double-stranded viral DNA (provirus). Step 3 . The provirus is integrated into the host cell genome producing ‘transformed host cell.’ Step 4. Integration of the provirus brings about replication of viral components which are then assembled and released by budding.

53. DNA Oncogenic Viruses. Virus Associated Tumour 1. PAPOVAVIRUSES Human papilloma virus Cervical cancer and its precursor lesions, squamous cell carcinoma at other sites Skin cancer in epidermodysplasia verruciformis Papillomas (warts) on skin, larynx, genitals (genital warts) HERPESVIRUSES Epstein-Barr virus Burkitt’s lymphoma Nasopharyngeal carcinoma Human herpesvirus (Kaposi's sarcoma herpesvirus ) Kaposi’s sarcoma Pleural effusion lymphoma POXVIRUSES Molluscum contagiosum , papilloma HEPADNAVIRUSES Hepatitis B virus Hepatocellular carcinoma

54. RNA Oncogenic Viruses. Virus Associated Tumour HUMAN T-CELL LYMPHOTROPIC VIRUS (HTLV) HTLV-I Adult T-cell leukaemia lymphoma (ATLL) HTLV-II T-cell variant of hairy cell leukaemia HEPATITIS C VIRUS HCV Hepatocellular carcinoma

55. FIELD CANCERIZATION “field cancerization ” was first introduced by Slaughter et al. in 1953 Braakhuis et al. (2003) Defined “The presence of one or more areas consisting of epithelial cells that have genetic alterations. A field lesion (or shortly ‘field’) has a monoclonal origin, and does not show invasive growth and metastatic behavior, the hallmark criteria of cancer.”

56. studying the presence of histologically abnormal tissue surrounding oral squamous cell carcinoma. Development of multiple primary tumors and locally recurrent cancer. Organ systems in which field cancerization has been described since then are: head and neck (oral cavity, oropharynx and larynx), lung, vulva, esophagus, cervix, breast, skin, colon and bladd er.

57. Genetically altered cells plays a central role. Initial phase - stem cell acquires genetic alterations and forms a “patch,” a clonal unit of altered daughter cells. Mutations in TP53 and have been reported for head and neck, lung, skin and breast cancer. Conversion of a patch into an expanding field is the next logical and critical step in epithelial carcinogenesis. (Additional genetic alteration are reqiured ) Proliferating field gradually displaces the normal mucosa.

58. In the mucosa of the head and neck & esophagus , such fields have been detected with dimensions of greater than 7 cm in diameter (not detected by routine diagnostic techniques ) leads to the development of one or more tumors within a contiguous field of preneoplastic cells fields often remain after surgery of the primary tumor lead to new cancers “ a second primary tumor ” or “ local recurrence ,” depending on the exact site and time interval

59. Three phenomena: • A wide field of aerodigestive mucosa that tends to be affected by pre-malignant disease • The frequent occurrence of multiple primary tumors in epithelial areas affected by widespread pre-malignant disease • The possibility of distant related primary tumors in the upper aerodigestive tract .

60. Implications for Therapy- Care should be taken about screening and directed biopsies in these patients. Frequent examination for high-risk patients. Clonal patches that are unable to be detected grossly and are beyond the initial scope of surgical excision including chemotherapy or radiotherapy

61. APPLIED ASPECT CHEMOTHERAPY TARGET CHEMOTHERAPY RADIOTHERAPY

62. CHEMOTHERAPY A chemotherapeutic agent affects a cell in its dividing cycle and induces irreparable damage to the DNA to induce cell death.

63. Classification according to phase-specific toxicity PHASE SPECIFIC CHEMOTHERAPY- Methotrexate - Antimetabolite that binds to dihydrofolate reductase preventing DNA synthesis in S-phase. (similar to the action of the ionizing radiations) Vinca alkaloids -preventing polymerization to form microtubules and is M-phase specific Vinblastine – arrest cell mitosis Kills proliferating cells during the cell cycle These synchronized cell enter into a phase of cell cycles that killed by the cytotoxic agents

64. CELL- CYCLE SPECIFIC CHEMOTHERAPY- Most chemotherapeutic agents are cell cycle-specific (cells that actively divides) Only proliferating cells remain fully sensitive to drug induced cytotoxicity To increase cell kill – increase the duration of exposure rather than increasing the drug dose CELLS CYCLE- NONSPECIFIC CHEMOTHERAPY- Alkylating agents & paltanium derivatives have an equal effect on tumour & normal cells whether they are proliferating or resting phase Linear dose response curve (greater the dose of drug grater the fractional cell kill)

65. TRAGETED SMALL MOLECULES AGAIST EPIDERMAL GROWTH FACTOR RECEPTORS Gifitinib & erlotinib are orally active (EGFR-TKI) epidermal growth factor receptors tyrosine kinase inhibitor Blocks EGFR signaling Inhibits- growth, proliferation & survival of many solid tumors Side effects of drug- acneiform rash

66. RADIATION THERAPY The ionizing radiations are known to induce damage to the nucleotide chains of DNA and induce cell death. When a single chain is damaged, the damage is repairable, but when the double stranded damage occurs the cell death results.

67. Oxygenated cells are more susceptible to radiation than hypoxic cells Cell death occurs with proliferation, therefore, rapidly growing tumors are more susceptible to injury. intrabony tumors are relatively radioresistant than the soft tissue tumors. CELL CYCLE & RADIATION INJURY- M Phase- mitosis very sensitive to radiation injury G1 phase- resting phase, moderately resistant S phase- DNA synthesis, moderately resistant to radiation G2 Phase- resting phase, sensitive G0 non cycling cells- moderate resistance

68. Radiation Strategies Shrinking fields: Selective radiation dosing to varying region depending on primary size and shape (e.g. 7,000 cGy to primary site as primary therapy, 5,000–5,500 cGy to N0 neck or adjuvant treatment) Brachytherapy : Delivery of radiation to malignant tissue by placement of permanent radioisotopes intraoperatively

69. Three-dimensional multiple treatment beam therapy: Utilizes CT and MRI imaging, multiple treatment fields arranged to maximize radiation dose to target area yet achieve maximum normal tissue sparing Hyperfractionated radiation therapy • Rationale : To allow normal cells to proliferate and to suppress the tumor cells in between the fractions. • Dose: 1 Gy twice daily, for total dose of 60 to 70 Gy over a period of 7 weeks.

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