1.  NON-lethal genetic damage  A tumor is formed by the clonal expansion of a single precursor cell (monoclonal)  Four classes of normal regulatory genes  PROTO-oncogenes  Oncogenes  Oncoproteins  DNA repair genes  Apoptosis genes  Carcinogenesis is a multistep process

2.  Carcinogenesis is a multistep process at both the phenotypic and the genetic levels.  It starts with a genetic damage:  Environmental  Chemical  Radiation  Viral  Inhereted

3.  Genetic damage lead to “ mutation”  single cell which has the genetic damage undergoes neoplastic prliferation ( clonal expansion) forming the tumor mass

4.  Self-sufficiency in growth signals  Insensitivity to growth-inhibiting signals  Evasion of apoptosis  Defects in DNA repair: “Spell checker”  Limitless replicative potential: Telomerase  Angiogenesis  Invasive ability  Metastatic ability

5.  Remember the cell cycle !!  Binding of a growth factor to its receptor on the cell membrane  Activation of the growth factor receptor leading to activation of signal-transducing proteins  Transmission of the signal to the nucleus  Induction of the DNA transcription  Entry in the cell cycle and cell division

6. 6 4. Nuclear Proteins: Transcription Factors 5. Cell Growth Genes 3. Cytoplasmic Signal Transduction Proteins 1. Secreted Growth Factors 2. Growth Factor Receptors Functions of Cellular Proto-Oncogenes

7. Regulation of G1/S cell cycle transition Cell cycle arrest at G1/S (in response to DNA damage or other stressors) is medicated through which gene? p53 (levels of p53 under negative regulation by MDM2 and p14 ARF)

8. INHIBITORS: Cip/Kip, INK4/ARF Tumor (really growth) suppressor genes: p53

9.  -cyclins are only expressed at specific stages of the cell cycle  -cyclin-dependent kinases are expressed constitutively, but must bind cyclins for  activation; phosphorylation of target proteins essential for progression through  cell cycle

10. 5- Cyclins and cyclins- dependent kinases (CDKs)  Progression of cells through cell cycles is regulated by CDKs after they are activated by binding with cyclins  Mutations that dysregulate cyclins and CDKs will lead to cell proliferation …e.g.  Cyclin D genes are overexpressed in breast, esophagus and liver cancers.  CDK4 is amplified in melanoma and sarcomas

11.  RB gene exists in “ active “ and “ inactive” forms  If active  will stop the advancing from G1 to S phase in cell cycle  If cell is stimulated by growth factors  inactivation of RB gene  brake is released  cells start cell cycle …G1  S  M …then RB gene is activated again

13. 13 Hanahan and Weinberg, Cell 100: 57, 2000 Apoptosis Oncogenes Tumor Suppressor Inv. and Mets Angiogenesis Cell cycle

14.  Four classes of normal regulatory genes  PROTO-oncogenes  Oncogenes  Oncoproteins  DNA repair genes  Apoptosis genes  Carcinogenesis is a multistep process

15.  Oncogenes are mutated forms of cellular proto-oncogenes.  Proto-oncogenes code for cellular proteins which regulate normal cell growth and differentiation. 15

16.  Are MUTATIONS of NORMAL genes (PROTO-oncogenes)  Growth Factors  Growth Factor Receptors  Signal Transduction Proteins (RAS)  Nuclear Regulatory Proteins  Cell Cycle Regulators  Oncogenes code for  Oncoproteins

17.  Oncogene  : a cancer-causing gene that has been mutated to cause an increase in  activity, or the activity becomes constitutive, or a new activity is acquired.  -a mutation in a single allele is sufficient to transform cells (dominant).  -originally identified as viral proteins that resembled normal human proteins.  -the term "proto-oncogene" refers to the normal protein that has not been mutated

18.  tumor Suppressor gene  Mutation of tumor suppressor gene cause a loss offunction.  -mutations are required in both alleles to transform cells (recessive)

19. 19 Class I: Growth Factors Class II: Receptors for Growth Factors and Hormones Class III: Intracellular Signal Transducers Class IV: Nuclear Transcription Factors Class V: Cell-Cycle Control Proteins Five types of proteins encoded by proto- oncogenes participate in control of cell growth:

21. 1 2 3 4 4 types of genetic mutations that contribute to cancer

22.  Categories of oncogenes  A. Growth factors  -generally not directly involved transformation, but increased expression seen as part of  an autocrine loop due to changes in other steps in the same pathway

24.  -They are transmembrane proteins with an external ligand binding domain and an  internal tyrsosine kinase domain.  -oncogenic mutations can result in dimerization and activation in the absence of  ligand  -more commonly, increased activity is a result of overexpression of receptors

25.  They are transmembrane proteins with an external ligand binding domain and an  internal tyrsosine kinase domain.  -Oncogenic mutations can result in dimerization and activation in the absence of  ligand  -More commonly, increased activity is a result of overexpression of receptors.

27.  -Activated directly or indirectly by growth factor receptors  -Activation of signal transducers triggers a phosporylation cascade that ultimately  results in changes in gene expression at the transcriptional level.  -mutations in RAS , a GTPase, are the most common oncogenic abnormality in tumors  -failure to hydrolyze GTP locks RAS in its active form.

28.  -Transcription factors contain DNA binding domains.  Sequences  Regulate expression of genes essential for passage through the cell cycle, or  regulation of apoptosis.  -

29. Category PROTO- Oncogene Mode of Activation Associated Human Tumor GFs PDGF-β chainSISOverexpressionAstrocytoma Osteosarcoma Fibroblast growth factors HST-1OverexpressionStomach cancer INT-2AmplificationBladder cancer Breast cancer Melanoma TGFα OverexpressionAstrocytomas Hepatocellular carcinomas HGF OverexpressionThyroid cancer

30. Category PROTO- Oncogene Mode of Activation Associated Human Tumor GF Receptors EGF-receptor family ERB-B1 (ECFR) OverexpressionSquamous cell carcinomas of lung, gliomas ERB-B2AmplificationBreast and ovarian cancers CSF-1 receptorFMSPoint mutationLeukemia Receptor for neurotrophic factors RETPoint mutationMultiple endocrine neoplasia 2A and B, familial medullary thyroid carcinomas PDGF receptorPDGF-ROverexpressionGliomas Receptor for stem cell (steel) factor KITPoint mutationGastrointestinal stromal tumors and other soft tissue tumors

31. Category PROTO- Oncogene Mode of Activation Associated Human Tumor Signal Transduction Proteins GTP-bindingK-RASPoint mutationColon, lung, and pancreatic tumors H-RASPoint mutationBladder and kidney tumors N-RASPoint mutationMelanomas, hematologic malignancies Nonreceptor tyrosine kinase ABLTranslocationChronic myeloid leukemia Acute lymphoblastic leukemia RAS signal transduction BRAFPoint mutationMelanomas WNT signal transduction β-cateninPoint mutationHepatoblastomas, hepatocellular carcinoma

32. Category PROTO- Oncogene Mode of Activation Associated Human Tumor Nuclear Regulatory Proteins Transcrip. activators C-MYCTranslocationBurkitt lymphoma N-MYCAmplificationNeuroblastoma, small cell carcinoma of lung L-MYCAmplificationSmall cell carcinoma of lung

33. Which signal transduction pathway is continuously activated by mutant RAS? MAP kinase pathway Point mutations of ras are seen in what % of all human malignancies? 15-20%

34.  It is a protooncogene  Found on Chromosome 8  Member of Myc protein family  Includes N-myc and L- myc  C-MYC

35.  Main functions:  Cell proliferation  Apoptosis

36.  The MYC protein can either activate or repress the transcription of other genes.  Activated by MYC are  growth-promoting genes, including cyclin dependent kinas (CDKs),  Genes repressed by MYC  THE CDK inhibitors (CDKIs)

37.  Dysregulation of MYC promotes TUMORIGENESIS by increasing expression of genes that promote proliferation in turn inactivates the inhibitors.

38.  c-Myc was first discovered in lymphoma patients  Reciprocal translocation from chromosome 8 to chromosome 14 http://www.ncbi.nlm.nih.gov/books/bv.fcgi?call=b v.View..ShowSection&rid=gnd.section.92 http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/B/Bu rkittLymphoma.html

39.  Rare but extremely aggressive cancer  Predominantly affects children in Southern Africa  Solid tumor of B lymphocytes  High tendency to spread to CNS, bone marrow, other blood elements http://www.brown.edu/Courses/Digital_Path/systemic_path/female/burkitt.htmlhttp://tmcr.usuhs.mil/tmcr/chapter41/clinical.htm

40.  Over-expressed in 70% of all human cancers  Translocated in 90% of all Burkitt’s lymphoma cases  90% of gynecological cancers  80% of breast cancers  70% of colon cancers  Contributes to more than 70,000 cancer deaths annually in the U.S.

41.  Translocation in Burkitt lymphoma, a B cell tumor. (t9:22)  Amplified in breast, colon, lung, and many other cancers;  Amplified in  N-MYC NEUROBLASTOMAS  L-MYC small cell cancers of lung.

42. . Tumor suppressor were originally identified as inherited mutations that confer a  predisposition to cancer (familial form).

43.  Inactivation of tumor suppressors can occur  Sporadically  -sequential inactivation of both alleles in somatic cells  You may hear the term  haploinsufficiency , which refers to inactivation of a single  allele contributing to malignancy.  -usually not the initiating event, but exacerbating.  Viral inactivation  -HPV expresses proteins that inhibit Rb and p53 function.

44.  RB gene  P53 gene  APC/Beta Catenin  INK4/ARF locus  TGF beta pathway  NF-1  NF-2  VHL  WT-1Caderins

45.  It is a tumor suppressor gene  It is located on chromosome 13  It regulates G1 /S transition phase.  It occurs in active hypophosphorylated and inactive hyperphosphorylaed state

46.  A Loss of RB function confers a predisposition to retinoblastoma.  occurs in both the  familial form (early onset) and sporadic form.

48.  Red reflex  Leukocoria

49.  Knudson, in 1974, proposed two-hit hypothesis, which in molecular terms can be stated as follows:  1. Two mutations ( hits) are required to produce retinoblastoma. Both of the normal  alleles of the RB locus must be inactivated (hence the two hits) for the development of retinoblastoma.

50.  2.In familial cases, children inherit one defective copy of the RB gene in the germ line; the other copy is normal. Retinoblastoma develops when the normal RB gene is  lost in retinoblasts as a result of somatic mutation.  3.In sporadic cases, both normal RB alleles are lost by somatic mutation in one of the retinoblasts.  The end result is the same: a retinal cell that has lost both of the  normal copies of the RB gene becomes cancerous.

51.  Why Retinoblastoma is autosomal dominant?  Retinoblastoma families only a single somatic mutation is required for expression of the disease,.

52.  In hereditary retinoblastoma, an affected child inherits one defective Rb allele together with one normal gene. This is heterozygous state.  It is not associated with changes in the retina because 50% of the Rb gene product is sufficient to prevent  the development of retinoblastoma.  If the remaining normal Rb allele is inactivated by deletion or mutation, the loss of its suppressor function leads to the appearance of a neoplasm. This genetic process is referred to as loss of heterozygosity.

53.  The importance of Rb lies in its regulation of  the G1/S checkpoint

54.  loss of normal cell cycle control leads to malignant transformation  the four key regulators of the cell cycle (CDKN2A, cyclin D,CDK4, Rb) is mutated in most human cancers.

55.  Human papillomavirus(HPV) produce E7 protein and the protein E7 binds to the hypophosphorylated form of Rb in place of E2F leading to uncontrolled growth.

56.  SPORADIC (Non- hereditary)  Unilateral, unifocal.  60% of all cases.  Present later.  Children of the affected are  normal.  Chromosomal anomaly is a  somatic mutation.  Relatives have a low risk of  RB development  FAMILIAL (Hereditary) 85% bilateral, multifocal. 40% % of all cases. Present earlier. Children of the affected have 45% chance of inheritance. Chromosomal anomaly is a Germline mutation. Autosomal dominant with high penetrance.

57.  The median age at presentation is 2 years, although the tumor may be present at birth.  Clinical features  poor vision, strabismus,  A whitish hue to the pupil ("cat's eye reflex"),  pain and tenderness in the eye.

58.  The TP53 gene which encodes p53 resides on the short arm of chromosome 17.

59.  In healthy unstressed cells,  p53 is short half life (20 min)  It undergo destruction by MDM2.

60.  IT HAS DIFFERENT NAMES  “GUARDIAN OF THE GENOME.”  GATE KEEPER. MOLECULAR POLICE MAN.

61.  When cell is stressed,  When ever there is a DNA Damage  ATM (ataxia telangiectasia mutated) are activated.  These activated complexes release P53 from  MDM2 and increase its half-life and enhance its ability to drive the transcription of target genes. Hundreds of genes whose transcription is triggered by p53 have been found.

62.  1.Temporary cell cycle arrest.  2.permanent cell cycle arrest  3.Triggering of programmed cell death (termed apoptosis).  4.p53 plays a central role in maintaining the integrity of the genome.

63.  P53 is activated by the following .DNA damage  by irradiation, chemicals, uv light and Free radicle injury and senescence.

64. Point mutations MDM2 –degrade P53 E6 protein-From HPV

65.  p53-mediated cell cycle arrest in response to DNA damage in  The late G1 phase and is caused mainly by p53- dependent  transcription of the CDKI gene CDKN1A (p21)belongs to KIP/CIP group of CDKI.

66.  P53 induces expression of DNA damage repair genes to reapir the damaged DNA.

67.  p53-induced apoptosis of cells with irreversible DNA damage is the ultimate protective mechanism against neoplastic transformation by pro-apoptotic genes such as BAX.

68.  70% of human cancers have a defect  in P53 gene,  Most commonly in Breast, colon and lung cancer

69.  Less commonly, some patients inherit a mutant TP53 allele The disease is called the  Li-Fraumeni syndrome.

70.  patients with Li- Fraumeni syndrome develop tumors at a younger age and may develop multiple primary tumors.

71.  25-fold greater chance of developing a malignant tumor by age 50 in a person with mutant single allele.  sarcomas, breast cancer, leukemia, brain tumors, and  carcinomas of the adrenal cortex.  patients with Li-Fraumeni  syndrome develop tumors at a younger age and may develop multiple primary tumors.

72. Normal function of p53 is to upregulate activity of which 2 genes to allow repair of DNA? p21 GADD45

73.  p53 inhibits G1 progression only in response to DNA damage  -normally p53 is very unstable, due to proteolytic degradation triggered by mdm2 .p53 is phosphorylated in response to DNA damage; mdm2 no longer binds p53  -p53 upregulates expression of p21, which in turn inhibits G1/S CDKs.  c. In response to excessive DNA damage, p53 can trigger apoptosis

74.  TGF- β  COLON  E-cadherin  STOMACH  NF-1,2  NEURAL TUMORS  APC/ β -cadherin  GI, MELANOMA  SMADs  GI  RB  RETINOBLASTOMA  P53  EVERYTHING!!  WT-1  WILMS TUMOR  p16 (INK4a)  GI, BREAST  BRCA-1,2  BREAST  KLF6  PROSTATE

75.  BCL-2  p53  MYC

76.  DNA repair is like a spell checker  HNPCC (Hereditary Non-Polyposis Colon Cancer [Lynch]): TGF- β, β -catenin, BAX  Xeroderma Pigmentosum: UV fixing gene  Ataxia Telangiectasia: ATM gene  Bloom Syndrome: defective helicase  Fanconi anemia

77.  TELOMERES determine the limited number of duplications a cell will have, like a cat with nine lives.  TELOMERASE, present in >90% of human cancers, changes telomeres so they will have UNLIMITED replicative potential

78.  Q : How close to a blood vessel must a cell be?  A: 1-2 mm  Activation of VEGF and FGF-b  Tumor size is regulated (allowed) by angiogenesis/anti-angiogenesis balance

80.  Detachment ("loosening up") of the tumor cells from each other  Attachment to matrix components  Degradation of ECM, e.g., collagenase, etc.  Migration of tumor cells

82.  NM23  KAI-1  KiSS

83.  TRANSLOCATIONS and INVERSIONS  Occur in MOST Lymphomas/Leukemias  Occur in MANY (and growing numbers) of NON-hematologic malignancies also

84. MalignancyTranslocationAffected Genes Chronic myeloid leukemia(9;22)(q34;q11)Ab1 9q34 bcr 22q11 Acute leukemias (AML and ALL)(4;11)(q21;q23)AF4 4q21 MLL 11q23 (6;11)(q27;q23)AF6 6q27 MLL 11q23 Burkitt lymphoma(8;14)(q24;q32)c-myc 8q24 IgH 14q32 Mantle cell lymphoma(11;14)(q13;q32)Cyclin D 11q13 IgH 14q32 Follicular lymphoma(14;18)(q32;q21)IgH 14q32 bcl-2 18q21 T-cell acute lymphoblastic leukemia(8;14)(q24;q11)c-myc 8q24 TCR-α 14q11 (10;14)(q24;q11)Hox 11 10q24 TCR-α 14q11 Ewing sarcoma(11;22)(q24;q12)Fl-1 11q24 EWS 22q12

85.  NO single oncogene causes cancer  BOTH several oncogenes AND several tumor suppressor genes must be involved  Gatekeeper/Caretaker concept  Gatekeepers: ONCOGENES and TUMOR SUPPRESSOR GENES  Caretakers: DNA REPAIR GENES  Tumor “PROGRESSION”  ANGIOGENESIS  HETEROGENEITY from original single cell

86.  Initiation/Promotion concept:  BOTH initiators AND promotors are needed  NEITHER can cause cancer by itself  INITIATORS (carcinogens) cause MUTATIONS  PROMOTORS are NOT carcinogenic by themselves, and MUST take effect AFTER initiation, NOT before  PROMOTORS enhance the proliferation of initiated cells

88.  Inflammation ?  Teratogenesis ?  Immune Suppression?  Neoplasia?  Mutations?

89.  1) Chemicals  2) Radiation  3) Infectious Pathogens

90.  DIRECT  β -Propiolactone  Dimeth. sulfate  Diepoxybutane  Anticancer drugs (cyclophosphamide, chlorambucil, nitrosoureas, and others)  Acylating Agents  1-Acetyl-imidazole  Dimethylcarbamyl chloride  “PRO”CARCINOGE NS  Polycyclic and Heterocyclic Aromatic Hydrocarbons  Aromatic Amines, Amides, Azo Dyes  Natural Plant and Microbial Products  Aflatoxin B1  Hepatomas  Griseofulvin  Antifungal  Cycasin  from cycads  Safrole  from sassafras  Betel nuts  Oral SCC

91.  OTHERS  Nitrosamine and amides (tar, nitrites)  Vinyl chloride  angiosarcoma in Kentucky  Nickel  Chromium  Insecticides  Fungicides  PolyChlorinated Biphenyls (PCBs)

92.  HORMONES  PHORBOL ESTERS (TPA), activate kinase C  PHENOLS  DRUGS, many “Initiated” cells respond and proliferate FASTER to promotors than normal cells

93.  UV: BCC, SCC, MM (i.e., all 3)  IONIZING: photons and particulate  Hematopoetic and Thyroid (90%/15yrs) tumors in fallout victims  Solid tumors either less susceptible or require a longer latency period than LEUK/LYMPH  BCCs in Therapeutic Radiation

94.  HPV  SCC  EBV  Burkitt Lymphoma  HBV  HepatoCellular Carcinoma (Hepatoma)  HTLV1  T-Cell Malignancies  KSHV  Kaposi Sarcoma

95.  100% of gastric lymphomas (i.e., M.A.L.T.-omas)  Gastric CARCINOMAS also!

96.  IMMUNE SURVEILLENCE CONCEPT  CD8+ T-Cells  NK cells  MACROPHAGES  ANTIBODIES

97.  Mutation, like microbes  ↓ MHC molecules on tumor cell surface  Lack of CO-stimulation molecules, e.g., (CD28, ICOS), not just Ag-Ab recognition  Immunosuppressive agents  Antigen masking  Apoptosis of cytotoxic T-Cells (CD8), i.e., the damn tumor cell KILLS the T-cell!

98.  Location  anatomic ENCROACHMENT  HORMONE production  Bleeding, Infection  ACUTE symptoms, e.g., rupture, infarction  METASTASES

99.  Reduced diet: Fat loss>Muscle loss  Cachexia: Fat loss AND Muscle loss  TNF ( α by default)  IL-(6)  PIF (Proteolysis Inducing Factor)

100.  Progressive weakness, loss of appetite, anemia and profound weight loss (>20%)  Often correlates with tumor mass & spread  Etiology includes a generalized increase in metabolism and central effects of tumor on hypothalamus  Probably related to macrophage production of TNF-a

101.  Due to Products released by tumor  Cushing’s Syndrome  Adrenal, Lung Ca – ACTH  Inappropriate ADH syndrome (Hyponatremia) – lung ca  Hypothalamic tumors (vasopressin)  Hypercalcemia – Ca is the common cause. – lung.  Hypoglycemia - insulin or insulin like activities Fibrosarcoma, Cerebellar hemangioma.

102.  Endocrine (next)  Nerve/Muscle, e.g., myasthenia w. lung ca.  Skin: e.g., acanthosis nigricans, dermatomyositis  Bone/Joint/Soft tissue: HPOA (Hypertrophic Pulmonary OsteoArthropathy)  Vascular: Trousseau, Endocarditis  Hematologic: Anemias  Renal: e.g., Nephrotic Syndrome

103. Cushing syndromeSmall cell carcinoma of lungACTH or ACTH-like substance Pancreatic carcinoma Neural tumors Syndrome of inappropriate antidiuretic hormone secretion Small cell carcinoma of lung; intracranial neoplasms Antidiuretic hormone or atrial natriuretic hormones HypercalcemiaSquamous cell carcinoma of lung Parathyroid hormone-related protein (PTHRP), TGF-α, TNF, IL-1 Breast carcinoma Renal carcinoma Adult T-cell leukemia/lymphoma Ovarian carcinoma HypoglycemiaFibrosarcomaInsulin or insulin-like substance Other mesenchymal sarcomas Hepatocellular carcinoma Carcinoid syndromeBronchial adenoma (carcinoid)Serotonin, bradykinin Pancreatic carcinoma Gastric carcinoma PolycythemiaRenal carcinomaErythropoietin Cerebellar hemangioma Hepatocellular carcinoma

104.  GRADING: HOW “DIFFERENTIATED” ARE THE CELLS?  STAGING: HOW MUCH ANATOMIC EXTENSION? TNM  Which one of the above do you think is more important?

105.  Grading – Cellular Differentiation (Microscopic)  Staging – Progression or Spread (clinical)

107. WELL? (pearls) MODERATE? (intercellular bridges) POOR? (WTF!?!) GRADING for Squamous Cell Carcinoma

109.  BIOPSY  CYTOLOGY: (exfoliative)  CYTOLOGY: (FNA, Fine Needle Aspirate)

110.  Categorization of undifferentiated tumors  Leukemias/Lymphomas  Site of origin  Receptors, e.g., ERA, PRA

130.  Prostatic Carcinoma-Bone  Lung Carcinoma-Adrenals & Brain  Neuroblastoma-Liver & Bone  Less common sites of metastases include  skin, muscle thyroid, breast….etc.

131.  CA LUNG-Smoking  CA CERVIX-Sexual transmission of HPV  CA BLADDER -Rubber Industry  CA LIVER --Aflatoxin & HBV infection  CA THYROID-Radiation  ANGIOSARCOMA of Liver-Plastic(PVC)  MESOTHELIOMA -Asbestos

132.  Location of tumor is of importance  1- Mass effect by pressing on vital areas  e.g. airway, intestine, BV, brain, nerve  obstruction, infarction, paralysis…etc  2- Local destruction of epithelial surface or BV  ulceration, bleeding, infection3- Hormonal activity

133.  Wasting syndrome characterized by anorexia, loss of body fat & weight, with marked weakness, anemia & fever.  Reduced food intake but high metabolic rate  Possibly due to release of cytokines by tumor cells & macrophages

134.  Systemic symptoms that can't be explained  by effects of local or distant spread of tumor or hormones appropriate to tumor tissue.  Due to ectopic production of hormones or other factors  They may precede the tumor or mimic metastases  They occur in about10%-15%of malignant tumors.

135.  History & clinical examination  Radiographic techniques  1- X ray  2CT scan  3- MRI  4-Ultrasound  5-Laboratory tests : general & specialized

136.  This is very important as many cancers a recurable if they are diagnosed early.  Specific symptoms should be followed upe.g. Abnormal bleeding Change of voice Change in a nevus Abnormal lump in breast  An ulcer that does not heal……etc. 

137.  Self examination of the breast- Mammography- Serial PAP smears for the cervix- Serial sputum cytology in smokers- Serial urine cytology in some cases, e.g. workers in rubber  Screening for genetic mutations in familial cancers.

138.  Study of cells :- Smear-  FNA, Brush, Fluid tapping…etc  Papanicolaou stain (PAP)often used.  False(+), False (-)- A negative report does not exclude malignancy, repeat- Advise biopsy, even if (+ )  1-Morphological Methods :

139.  Biopsy of tissue: Needle & core biopsy, Endoscopic Biopsy, or open surgical biopsy  Frozen Section (Rapid technique)  Paraffin Section ( 36-48 hrs. or longer )  H&E, Special histochemical stains stains) or by IMMUNOHISTOCHEMICALMethods

140.  Tumor markers represent biochemical indicators of the presence of a tumor.  Their uses are to I - Confirm diagnosis.  II -Determine the response to treatment.  III - Detect early relapse.  Present in serum or urine.  Many are present in normal & tumor tissue, so they are not very specific but their level is important.

141.  Hormones  Human Chorionic Gonadotrophic Hormone(  HCG)Elevated levels are seen in Pregnancy& Gestational Trophoblastic Disease  Calcitonin useful in diagnosis of some thyroid carcinomas 

142.  Carcinoembryonic Antigen ( CEA ) : in fetal tissue & some malignancies  Colorectal CA & Pancreatic CA  Alpha Fetoprotein (AFP):Cirrhosis : Elevated Hepatocellular carcinoma : Extremely high

143.  Prostatic Acid Phosphatase ( PAP )  levels seen in Metastatic prostatic CA Useful in : Staging prostatic CA  Assessment of prognosis Response to therapy.

144.  MUC-1 in breast CA  CA-125 in ovarian CA  CA-19-9 in pancreatic & hepatobiliary CA