TRANSFORMATION & PROGRESSION Self-sufficiency in growth
signals Insensitivity to growth-inhibiting signals Evasion of
apoptosis Limitless replicative potential: Telomerase Angiogenesis
Invasive ability Metastatic ability Defects in DNA repair: Spell
checker
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Evasion of APOPTOSIS BCL-2 Overexpression Seen in B-cell
lymphomas of follicular type t(14;18)(q32;q21),dominant with IgH
genes and overexpression of BCL-2 protein P53 (Pro-apoptic gene) -
Directly activates Bax (Bcl-2 protein) Reduced levels of CD95/Fas
FLIP protein Inhibits Caspase 8 Loss of APAF1(Apoptosis activating
factor-1) Increased IAP (inhibitors of apoptosis)
Slide 6
LIMITLESS REPLICATIVE POTENTIAL TELOMERES determine the limited
number of duplications a cell will have! TELOMERASE, present in
>90% of human cancers, changes telomeres so they will have
UNLIMITED replicative potential
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TUMOR ANGIOGENESIS 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
Slide 9
Proteases of tumors can induce production of angiogenic factors
and inhibitors Proteases act on ECM components (E.g. Collagen)
releasing bFGF and VEGF VEGF triggers the NOTCH Pathway
Slide 10
INVADE & METASTASIS 1. Invasion of Extracellular Matrix 2.
Vascular Dissemination Invading the Extracellular matrix requires
breaching the basement membrane Intersitial Connective Tissue
Vascular Basement Membrane
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Invasion of ECM 1. Detachment ("loosening up") of the tumor
cells from each other Down-regulation of E-cadherin and
Beta-catenin reducing cells adherance to each other
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Invasion of ECM 2. Degradation of ECM, e.g., collagenase, etc.
Break down the basement membrane Proteases MMP (matrix
metalloproteinases) Releasing growth factors (e.g. VEGF) and
angiogenic, growth promoting effects with cleavage of Type IV
collagen for example 3. Attachment to matrix components Breakdown
by MMP allows tumor cell receptors to bind ECM proteins and
stimulate migration E.g. MMP2 cleaves laminin and MMP9 cleaves
collagen IV Change in tumor cell receptor attachment to ECM
proteins Stimulates Migration
Slide 13
Invasion of ECM Migration of tumor cells Cytokines ( autocrine
factors) Cleavage of the ECM components (Laminin, collagen) and
growth factors (IGF I, II) that have chemotactic activity Stromal
cells around tumor also produce hepatocyte growth factor-scatter
factor promoting motility
Vascular Dissemination Following intravasation, clumps of
platelet-tumor aggregates are formed Extravastion of this tumor
emboli at distant sites Attachment to the endothelium Exits through
the basement membrane Followed by migration to lymphoid tissue
using the CD44 adhesion molecule (expressed on T cells) CD44 binds
to hyaluronate allowing migration and towards metastic spread *Most
metastases occur in the first capillary bed available to tumor
Slide 17
Other possible mechanisms explaining metastasis: 1. Tumor cell
adhesion molecule ligands are expressed on the endothelial cell of
target tissue Target organ variation in endothelial cell expression
of adhesion molecule ligands 2. Chemokine receptors are expressed
in cancer cells that allowing binding of chemokines expressed at
specific tissues E.g. CXCR4 and CCR7 receptors expressed in breast
cancer cells 3. Chemoattractants E.g. IGF 1, II, can be expressed
at target tissues
Slide 18
Breast cancer cells, how do they adapt and make their
environment habitable? - Osteolytic behavior in bone by osteoclasts
Tumor cells PTHRP (parathyroid hormone-related protein) Osteoblasts
RANK ligand Osteoclasts Osteoclasts break down the matrix releasing
growth factors
Slide 19
Development of Metastasis One hypothesis suggest why only some
tumors metastasize? Metastasis Signature Multiple abnormalities
taking place in the early stage of development Intrinsic
properties, microenvironment (stroma), angiogenesis, resistance to
immune defenders, and local invasiveness Other hypothesis Rare
clonal variation that develop metastasis
Slide 20
DEFECTS IN DNA REPAIR 1. Mismatch Repair 2. Nucleotide Excision
Repair 3. Recombination Repair
Slide 21
DNA REPAIR GENE DEFECTS DNA repair is like a spell checker
HNPCC (Hereditary Non-Polyposis Colon Cancer [Lynch]): TGF- , -
catenin, BAX Xeroderma Pigmentosum: UV fixing gene Ataxia
Telangiectasia: ATM gene Bloom Syndrome: defective helicase Fanconi
anemia
Slide 22
Hereditary Non-Polyposis Colon Cancer Mismatch Repair
Proofreaders disabled Microsatellite fragments 1 to 6 nucleotide
repeats Creates instability Dna mismatch repair gene affect cell
growth indirectly, through mutations in other genes TGF-beta,
beta-catenin, Bax
Slide 23
Xeroderma Pigmentosum UV radiation exposure Pyrimidine dimers
DNA damage with defect in nucleotide excision repair
Slide 24
Autosomal Recessive Disorders Defect in DNA repair by
Homologous Recombination Ataxia Telangiactasia neurological
degenerative disorder, exposure to Ionizing radiation Defective ATM
gene Bloom Syndrome - Ionizing radiation resulting in developmental
defects Defect in gene of chromosme 15 encodes helicase dna repair
through homologus recombination Fanconi anemia Chemotherapeutic
agents causing damage Bone-marrow aplasia BRCA1 and BRCA 2 Proteins
take part in homologous recombination repair pathway Mutation of
BRCA2 gene chromosomal breaks & aneuploidy
Slide 25
STROMAL MICROENVIRONMENT Cross-Talk between the tumor cells and
ECM E.g. Laminin degradation by MMP2 Stimulates tumor cell motility
E.g. Type IV collagen degradation by MMP9 Release of VEGF Storage
of inactive growth factors in ECM, proteases release the active
form (E.g. PDGF, TGF-beta, bFGF)
Slide 26
STROMAL MICROENVIRONMENT Inflammatory Cells and fibroblasts
Cytokines released during inflammation Induce survival and
progression of tumor cells Chemokines Chemotactic properties
Fibroblasts Important role in tumor cells Desmoplastic response,
highly fibrotic, promoting growth
Slide 27
Metabolism of Tumors
Slide 28
WARBURG EFFECT Otto Warburg, Nobel Prize discovery on the
metabolism of turmors Cancer cells rely on Aerobic Glycolysis,
Warburg Effect, instead of the mitochondrial oxidative
phosphorylation Several Hypothesis 1. During angiogenesis, vessels
are poorly formed, activation of H1F1alpha due to hypoxia increases
expression of enzymes of glycolysis, and vasculature 2. Exposure to
state of continous hypoxia and normoxia 3. Mutations in oncogene
and tumor supressors (p53, RAS, PTEN) stimulate metabolic
changes
Slide 29
Metabolic Alterations In addition, tumor cells need to build
and maintain its membrane, proteins, and organelles --- Anabolic
Pathway Lipid, nucleotide, proteins (amino acids), glucose (carbon)
Glutamine Both glycolytic and anabolic pathway
Slide 30
PI3K/AKT/Mtor pathway Important in regulating normal cell
functions including amino acid, glucose uptake In tumors, mutation
of tumor supressors and oncogenes - -- Abnormality in pathway can
occur favoring survival and proliferation of tumor cells E.g. PTEN
has a negative regulation of this pathway,
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Other tumor supressors that supress this pathway:- Peutz-Jegher
syndrome mutated gene LKB1 ( Liver kinase B1) Limits or regulates
cell growth by AMP-dependent protein kinase mechanism Negative
regulator of mTOR pathway Supression of LKBI (also called STK11)
Increase in anabolic metabolism of cancer cells TuberousSclerosis
mutated genes (TSC1 and TSC2) ----- Negative regulation of
mTOR
Slide 33
CHROMOSOME CHANGES in CANCER TRANSLOCATIONS and INVERSIONS
Occur in MOST Lymphomas/Leukemias Occur in MANY (and growing
numbers) of NON-hematologic malignancies also
Translocations ------------- Activate Proto-Oncogenes 2 common
mechanism of translocation: A) Lymphoid Tumors --- Swapping or
movement of segment of chromosome to another chromosome B)
Hematopoietic tumors --- Two chromosomes recombine to form hybrid
fusion genes for production of chimeric proteins
Slide 36
Burkitt Lymphoma:- t(8:14) (q24;q32) Translocation at
chromosome 8 (c-MYC) and 14 ( IGH gene) MYC containing segment of
chromosome 8 movement to to chromosome 14q32 at IGH gene
Overexpression of MYC
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Follicular Lymphomas:- t(14:18) (q32:q21) Translocation at
chromosome 14 (IGH gene) and 18 (BCL2) Activation of BCL2
Slide 39
Chronic Myeloid Leukemia (CML):- t(9;22) (q34;q11)
Translocation at chromosome 9 (ABL gene) and 22 (BCR gene)
Slide 40
What is the Philadelphia chromosome? Chromosome 22 that
exchanges portion (reciprocal translocation) of the chromosome with
Chromosome 9 Fusion of two genes BCR-ABL producing protein with
tyrosine kinase activity Found in both CML & ALL (Acute
Lymphoblastic Leukemia)
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Ewing Sarcoma Also known as PNET (Primitive neuroectodermal
tumor) T(11:22) (q24;q12) FLI 1 gene and EWSR1 gene Chimeric
protein EWS-FLI 1 Fusion of most genes encode for transcription
factors
Slide 43
GENE AMPLIFICATION Amplification of particular chromosomal
regions that contain the oncogenic sequence Hallmark Features:-
Double minutes ---- Small, centric structures or chromatin bodies
of extrachromosomal dna Homogenous staining regions Once a gene is
amplified, these amplified regions can be inserted into new
choromsome Lack a normal banding pattern and appear homogenous
(G-banded karyotype)
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Gene Amplification N-MYC ---- Neuroblastoma ERBB2 --- Breast
Cancers C-myc, L-myc, N-myc --- Small cell lung cancer **Greater
the amplification, poorer the prognosis
Slide 46
Epigenetic Changes Heritable changes in gene expression without
changing the DNA sequences Post-translational modfications of
histones + DNA methylation ===== Gene Expression Cancer cells ---
Hypermethylation within promoter regions and global DNA
Hypomethylation Tumor supressor genes --- hypermethlation ---
Silenced E.g. BRCA1, VHL, MLH1
Slide 47
Epigenetic Changes Genomic Imprinting: Maternal or paternal
allele of a gene or chromosome is modified by methylation and
inactivated
Slide 48
miRNAs Non-coding, single stranded RNA RNA-induced silencing
complex Post-transcriptional gene silencing Downregulate gene
expression by translational repression, and deadnylation In cancer
cells, increase oncogene expression or reduce tumor supression gene
expression
Slide 49
miRNAs 1. Reduced miRNA (Inhibitor of oncogene translation) ---
------------ Overproductin of oncogene 2. miRNA reduces tumor
supressor gene
Slide 50
Molecular Basis of Carcinogenesis
Slide 51
Carcinogenesis is MULTISTEP 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
Slide 52
Cancer result from accumulation of multiple mutations
Slide 53
Chimney Sweeps ---------- Scrotal skin cancer
Slide 54
Two-stage experimental model (mouse skin) of cancer development
(Skin Cancer):- Initiation ---- Exposure of cells to a sufficient
dose of a carcinogenic agent (Initiator) Promoter ----- Induce
tumors in Initiated cells
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Carcinogenesis: The USUAL (3) Suspects 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 PROMOTERS enhance the proliferation of
initiated cells Promoters are non-tumorigenic by themselves
Slide 56
Initiators === Highly reactive electrophiles ( Electron
deficient) atoms Bind to nucleophiles ( Electron rich sites) in
cell E.g. DNA, RNA, Proteins
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Q: WHO are the usual suspects? Inflammation ? Teratogenesis ?
Immune Suppression? Neoplasia? Mutations?
Slide 59
A: The SAME 3 that are ALWAYS blamed! 1) Chemicals 2) Radiation
3) Infectious Pathogens
Slide 60
CHEMICAL CARCINOGENS: INITIATORS DIRECT -Propiolactone Dimeth.
sulfate Diepoxybutane Anticancer drugs (cyclophosphamide,
chlorambucil, nitrosoureas, and others) Acylating Agents
1-Acetyl-imidazole Dimethylcarbamyl chloride PROCARCINOGEN S
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
Slide 61
CHEMICAL CARCINOGENS: INITIATORS OTHERS Nitrosamine and amides
(tar, nitrites) Vinyl chloride angiosarcoma in Kentucky Nickel
Chromium Insecticides Fungicides PolyChlorinated Biphenyls
(PCBs)
Slide 62
Cytochrome P450-dependent mono-xoygenase ----- Metabolizing
most carcinogens (E.g. Benzo [a]pyrene) ** Genes for these CP450
mono-oxygenase enzymes are polymorphic, determining susceptibility
to cancer E.g. CYP1A1 gene --- increases lung cancer in smokers
Carcinogen Aflatoxin B1 --- Mutation in p53 (transversion in codon
249 G:C T:A) or 249 (ser)
Slide 63
Nitrosamine in Food --Curing meat as a food preservation
Aflatoxin contamination
Slide 64
CHEMICAL CARCINOGENS: PROMOTORS HORMONES PHORBOL ESTERS (TPA),
activate kinase C PHENOLS DRUGS, many Initiated cells respond and
proliferate FASTER to promotors than normal cells
Slide 65
Following initiation, damaged dna template must be replicated
for the change to be heritable Promoter induction leads to
proliferation and clonal expansion of initiated cells
Slide 66
RADIATION CARCINOGENS UV: BCC, SCC, MM (i.e., all 3) IONIZING:
photons and particulate Hematopoetic and Thyroid (90%/15yrs) tumors
in fallout victims AML CML Solid tumors either less susceptible or
require a longer latency period than LEUK/LYMPH BCCs in Therapeutic
Radiation
Slide 67
Ultraviolet Rays UVA (320-400nm) UVB (280-320nm) -- Induction
of cutaneous cancers Pyrimidine dimers UVC (200-280nm) --- Filtered
by Ozone shield
Oncogenic RNA Virus Human T-Cell Leukemia Virus Type 1:- T-cell
leukemia / lymphoma Target -- CD4+ Tcells Induction of
Leukemogenesis In addition to the genome gag, pol, env, another
type tax gene is crucial Tax --- Viral replication, viral mrna
transcription, transcription of host cell genes for T-cell
differentiation Tax protein inactivation of Inhibitor p16/INK4a,
allowing increased cyclin D cell cycle dysregulation Proliferating
T Cells --- Mutations and genomic instability
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Oncogenic DNA Virus Human Papilloma Virus DNA Virus ---- 70
different types of HPV Low Risk type (E.g. HPV 6 & 11) Benign
squamous papillomas ( Warts) E.g. Genital area Episomal form
(non-integrated) High Risk type (E.g. HPV 16, 18, 31) Squamous cell
carcinoma of cervix Integrated form Genomic instability
Epstein-Barr Virus Commonly found in HIV patients,
immunocompromised patients Central Africa B-lymphocyte infection by
its CD21 receptor (attachment) Latent infection, no viral
replication Hijaciking of normal signal pathways Borrows a normal
b- cell activation pathway Latent membane protein (LBP-1) and
EBNA-2 --- oncogenic --- B-cell proliferation ---inducing b-cell
lymphomas
Slide 74
Epstein-Barr Virus EBV virus -------------- Burkitt Lymphoma??
Repeated infections such as malaria----small number of EBV infected
B cells persist ----- Lymphomas ------ Translocation ===== >
c-myc oncogene
Slide 75
HBV & HCV Integration into host cell explained by
immunologic response --- chronic inflammation (proliferation of
hepatocytes, growth factors, chemokines, cytokines) Activation of
NF-kB, blocks apoptosis Productin of ROS (reactive oxygen species)
HBx gene found in HBV ----- activates transcription factors **NO
viral oncoproteins
Slide 76
H. Pylori Carcinogenesis Similar to HBV, HCV, chronic
inflammation such as epithelial cell proliferation leads to gastric
adenocarcinoma Chronic gastritis Gastric atrophy Intestinal
metaplasia Dysplasia Cancer CagA (cytotoxin-associated A) gene
Signaling cascade Gastric Lymphoma (B-cell type) 100% of gastric
lymphomas (i.e., M.A.L.T.-omas), mucosa-
associated-lymphoid-tissue, similar features of normal Peyers
patches Gastric CARCINOMAS also!
Escape from Immune Surveillance 1. Selective outgrowth Strong
immunogenic subclones are eliminated 2. Loss of expression of MHC
molecules 3. Lack of costimulation (modulate immune response by T
and B cells) 4. Immunosupression Chemicals, Radiation, tumor
products TGF-beta 5. Antigen masking Hidden antigens by glycocalyx
molecule 6. Apoptosis of cytotoxic T cells By expressing FasL on
T-lymphocytes
Slide 79
CYTOTOXIC CD8+ T-CELLS are the main eliminators of tumor
cells
Slide 80
How do tumor cells escape immune surveillance? 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!
Slide 81
Clinical Features of Neoplasia
Slide 82
Effects of TUMOR on the HOST Location anatomic ENCROACHMENT
HORMONE production Bleeding, Infection ACUTE symptoms, e.g.,
rupture, infarction METASTASES
Slide 83
Local / Hormonal Effects Location E.g. Pituitary Adenoma ---
Hypopituitarism Endocrine Gland Neoplasm -- Endocrine Insufficiency
GIT Neoplasm --- Obstruction, Intussception or Telescoping, melena,
hematuria Hormone Production Mostly benign Eg. Adenoma of
pancreatic islets (beta cell) Increased insulin --- Hypoglycemia
E.g. Non-endocrine tumors ----- Paraneoplastic syndrome
Slide 84
CACHEXIA Progressive body fat loss & lean muscle, along
with profound weakness, anorexia, and anemia **Food intake reduced,
however basal metabolic rate is increased --- Why?? Cytokines:- TNF
( by default) IL-(6) PIF (Proteolysis Inducing Factor)
Slide 85
Cancer Cachexia 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
Slide 86
Paraneoplastic Syndromes Group of signs and symptoms as a
result of substance produced by a tumor or reaction to a tumor Not
related to growth, spread or metastasis of the tumor itself
Earliest manifestation or appearance of the paraneoplastic syndrome
may precede the diagnosis and give clue to its presence Mimic
metastic disease
Slide 87
Paraneoplastic Syndromes Due to Products released by tumor
(Ectopic Hormone production) Cushings Syndrome Adrenal, Small Cell
Lung Ca ACTH (Increased Corticotropin and pro-opiomelanocortin,
POMC) Hypercalcemia Cancer osteolysis is the common cause either
due to primary bone or metastasis Humoral hypercalcemia --
Parathyroid hormone- related protein (PTHRP production) Breast
(PTHRP Osteolytic), Squamous cell bronchogenic carcinoma
Slide 88
Paraneoplastic Syndromes Neuromyopathic paraneoplastic
syndromes Myasthenia Gravis --- Bronchogenic, Breast ca (Antibodies
against tumor antigens) Inappropriate ADH syndrome (Hyponatremia)
Small cell lung ca Acanthosis Nigricans -- Hyperkeratosis of skin
Hypertrophic Osteoarthropathy Hypothalamic tumors (vasopressin)
Hypoglycemia - insulin or insulin like activities in Fibrosarcoma,
Cerebellar hemangioma. Vascular and Hematologic Manifestations --
Migratory thrombophlebitis (Trousseau syndrome) --- Pancreatic
Ca
ENDOCRINE 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-, 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
Slide 91
Grading & Staging of Tumors Assess extent and spread of
neoplasm Making prognosis, and for treatment
Slide 92
GRADING/STAGING GRADING: HOW DIFFERENTIATED ARE THE CELLS?
STAGING: HOW MUCH ANATOMIC EXTENSION? TNM Which one of the above do
you think is more important?
Slide 93
Grading & Staging of Tumor Grading Cellular Differentiation
(Microscopic) Staging Progression or Spread (clinical)
Slide 94
Grading Degree of differentiation Number of mitoses
Architectural features Staging -------- TNM Size of lesion (T)
Extent of spread to lymph nodes (N) Presence or absence of
Metastases (M)
Excision:- Selection of area for excising large mass can be
challenging (center can be necrotic, periphery not reliable)
Preservation with caution --- Immersion in formalin Fixative for
Electron microscopy Refrigeration at optimal temperature for
molecular analysis
Slide 100
Fine needle aspiration:- Aspirating cells with small-bore
needle Cytologic examination of the smear Commonly breast, thyroid,
and lymph nodes Less Invasive and immediate need biopsies obtained,
BUT difficult Cytologic smears (PAP):- Smear, Fix, and Stain
Cervix, endometrial, bronchogenic, prostati for tumor cells in
abdominal, pleural joint and CSF Provides recognition of
differentiation to normal, dysplasia, malignant cells, cellular
changes specific to carcinoma in situ
Slide 101
IMMUNOHISTOCHEMISTRY Categorization of undifferentiated tumors
Antibodies specific to intermediate filaments 9E.g. cytokeratins,
desmin) Site of origin Organ-specific antigens E.g. PSA Prognostic
or therapeutic signficant molecules Receptors, e.g.,
Estrogen/progesterone receptors Protein products of oncogenes E.g.
ERBB2
Slide 102
Flow Cytometry:- Measure individual cell characteristics E.g.
DNA content of tumor cells T and B lymphocytes tumors Molecular
Diagnosis:- A) Diagnosis of NeoplasmsPolyclonal and monoclonal
proliferations of T or B Cells PCR FISH Hematopoietic neoplasms
(e.g. translocations) B) Prognosis of malignant neoplasms C)
Diagnosis of hereditary predisposition D) Detection of minimal
residual disease