recent advances in histopathology .> 21

1. MODERATOR : Prof. PARAMESHA
PRESENTER : Dr. ASHWINI K.T

2. NEOPLASIA
 Neo + Plasia  New + Growth.
 Tumour  Swelling  any swelling*
 Willis definition:
“A neoplasm is an abnormal mass of tissue, the growth of
which exceeds and is uncoordinated with that of the
normal tissue and persists in the same manner after
cessation of the stimuli which evoked the change”
 “Cell division without control”
 Irreversible DNA damage, resulting in autonomous
growth of abnormal cells

3. Cell population / Growth Control:
• Proliferation  Differentiation  Apoptosis * Normal
Stem cell
Apoptosis
Organ / Tissue
Neoplasia
Benign - Malignant
Carcinogen
Initiator + Promotor
Inflam
1. Proto-Oncogenes (growth factors)
2.Growth/Tumor suppressor genes.
3. Genes controlling Apoptosis.
4.Genes controlling DNA Repair.
RAS
Rb
MYC
p53

4. Non neoplastic Neoplastic
(Polyclonal) (Monoclonal)
Growth Disorders:
Normal Adaptation Benign Malignant
Hyperplasia
Hypertrophy
Aplasia
Atrophy
Metaplasia
Dysplasia

5. Cell division Control - Carcinogenesis*

6. Cancer Biology:
 Structure:
 Parenchyma – Neoplastic cells.
 Stroma: Non neoplastic - normal DNA
 Features:
 Differentiation – Maturation of cells.
 Rate of Growth – Mitotic rate / Ki 67
 Local invasion – Hemorrhage, necrosis, destruction
 Metastasis – Distant Spread.
6

7. • Tumors are clonal (one parent)
• But have different mutations  different
shapes & features.
• Each new mutation adds a new feature.
Malignant cells
Normal
cell
Fourth or
later mutation
Third
mutation
Second
mutation
First
mutation
More new mutations with time.

8. Features of Cancer cells:
Clinically each patient’s
cancer has a different mix
of features depending on
quality & quantity of
mutations
Changes With Time...!

10. Invasion and metastasis are biologic
hallmarks of malignant tumors
 For tumor cells to break loose from a primary
mass, enter blood vessels or lymphatics, and
produce a secondary growth at a distant site, they
must go through a series of step (the metastatic
cascade):
Divided into two phases:
1. Invasion of the extracellular matrix (ECM)
2. Vascular dissemination, homing of tumor cells,
and colonization

11. Metastasis

12. In situ carcinoma

13. Microinvasive

14. Vascular invasion

15. Metastatic deposit in brain

16. The metastatic cascade
Sequential steps involved in
the hematogenous spread of
a tumor

17. Invasion of Extracellular Matrix
Two types of ECM:
1. Basement membrane (BM) and
2. Interstitial connective tissue
Composition: ECM is made up of collagens, glycoproteins, and
proteoglycans
1. A carcinoma must first breach the underlying BM
2. Then traverse the interstitial connective tissue, and
3. Gain access to the circulation by penetrating the vascular
BM
This process is repeated in reverse when tumor cell emboli
extravasate at a distant site

18. Invasion of Extracellular Matrix
Invasion of the ECM initiates the metastatic cascade and
is an active process that can be resolved into several
steps
1. Changes (“loosening up”) of tumor cell-cell
interactions
2. Degradation of ECM
3. Attachment to novel ECM components
4. Migration of tumor cells

19. Sequence of events in the invasion of
epithelial basement membranes by
tumor cells:
Tumor cells detach from each other because of
reduced adhesiveness,
then secrete proteolytic enzymes, degrading the
basement membrane.
Binding to proteolytically generated binding sites
and
tumor cell migration follow

20. Invasion of Extracellular Matrix
Invasion of the ECM initiates the metastatic cascade and
is an active process that can be resolved into several
steps
1. Changes (“loosening up”) of tumor cell-cell
interactions
2. Degradation of ECM
3. Attachment to novel ECM components
4. Migration of tumor cells

21. 1-Dissociation of cells from one another
(“loosening up”) of tumor cell-cell interactions
As a result of alterations in intercellular adhesion
molecules

22. Normal cells are bound together by
adhesion molecules
 Cell-cell interactions are mediated by the cadherin
family of transmembrane glycoproteins
 Intracellularly the E-cadherins are connected to β-
catenin and the actin cytoskeleton

24. Tumors with down-regulated
E-cadherin expression
Seen in several epithelial tumors, including
adenocarcinomas of the colon and breast
 This down-regulation reduces the ability of cells to
adhere to each other and facilitates their
detachment from the primary tumor
 The normal function of E-cadherin is dependent
on its linkage to catenins
 In some tumors E-cadherin is normal, but its
expression is reduced because of mutations in the
gene for α catenin

26. Invasion of Extracellular Matrix
Invasion of the ECM initiates the metastatic cascade and
is an active process that can be resolved into several
steps
1. Changes (“loosening up”) of tumor cell-cell
interactions
2. Degradation of ECM
3. Attachment to novel ECM components
4. Migration of tumor cells

27. 2-Local degradation of the basement membrane
and interstitial connective tissue
Elaboration of proteases by
 Tumor cells themselves or
 Stromal cells [induced by tumor cells]
Many different families of proteases
 Matrix metalloproteinases (MMPs)
 Cathepsin D &
 Urokinase plasminogen activator

28. MMPs
Tumors either elaborate large quantities of MMPs or they
may reduce the concentrations of MMP-inhibitors
They regulate tumor invasion by:
 Dissolving components of the BM & interstitial matrix
 Releasing ECM-sequestered growth factors
 Cleavage products of collagen and proteoglycans also have
chemotactic, angiogenic, and growth-promoting effects
 Eg: MMP9 is a gelatinase that cleaves type IV collagen of the epithelial
and vascular basement membrane and also stimulates release of VEGF
from ECM-sequestered pools
Eg: Benign tumors of the breast, colon, and stomach show
little type IV collagenase activity, whereas their malignant
counterparts overexpress this enzyme

29. Invasion of Extracellular Matrix
Invasion of the ECM initiates the metastatic cascade and
is an active process that can be resolved into several
steps
1. Changes (“loosening up”) of tumor cell-cell
interactions
2. Degradation of ECM
3. Attachment to novel ECM components
4. Migration of tumor cells

30. 3 - Attachment to novel ECM components
 Normal epithelial cells have receptors, such as integrins, for
basement membrane laminin and collagens that are
polarized at their basal surface
 These receptors help to maintain the cells in a resting,
differentiated state
 Loss of adhesion in normal cells leads to induction of
apoptosis [tumor cells are resistant to this form of cell
death]
 The matrix itself is modified in ways that promote invasion
and metastasis
 Eg: cleavage of the basement membrane proteins collagen IV and
laminin by MMP2 or MMP9 generates novel sites that bind to
receptors on tumor cells and stimulate migration

31. Invasion of Extracellular Matrix
Invasion of the ECM initiates the metastatic cascade and
is an active process that can be resolved into several
steps
1. Changes (“loosening up”) of tumor cell-cell
interactions
2. Degradation of ECM
3. Attachment to novel ECM components
4. Migration of tumor cells

32. 4 – Migration of tumor cells - Locomotion
 Tumor cells propel themselves through the degraded basement
membranes and zones of matrix proteolysis
 It involves many families of receptors and signaling proteins that
eventually impinge on the actin cytoskeleton
 Cells must attach to the matrix at the leading edge, detach from the matrix
at the trailing edge, and contract the actin cytoskeleton to ratchet forward -
Ameboid migration
 Such movement are potentiated by tumor cell–derived cytokines
 Cleavage products of matrix components (e.g., collagen, laminin) and some
growth factors (e.g., IGFs I and II) have chemotactic activity for tumor cells
 Stromal cells also produce paracrine effectors of cell motility
 HGF–scatter factor, which bind to receptors on tumor cells
 HGF–scatter factor is elevated at the advancing edges of the highly invasive
brain tumor glioblastoma multiforme

33. Ameboid migration
 In this type of migration the cell squeezes through
spaces in the matrix instead of cutting its way through
it
 This ameboid migration is much quicker
 Tumor cells are capable of switching between the two
forms of migration, perhaps explaining the
disappointing performance of MMP inhibitors in
clinical trials

34. Malignant tumors have varied
metastatic potential
Cancer without metastatic potential
 Basal cell carcinoma
Cancer with high malignant potential
 Malignant melanoma
 Why this variation?
 What genetic changes bring about metastatic
potential?

35. Several THEORIES have been proposed to explain how
the metastatic phenotype arises?
1. The clonal evolution model
 As mutations accumulate in cancer cells, the tumor become
heterogeneous

36. 2. Metastasis is the result of multiple abnormalities that
occur in most of the cells in a primary tumor
 “Metastasis signature”
 It may involve the cancer cells or in the microenvironment
3. Background genetic variation in gene expression
contributes to the generation of metastases
4. Tumors derive from rare tumor stem cells, metastases
require the spread of the tumor stem cells
themselves

37. Mechanisms of metastasis
development within a
primary tumor:
A nonmetastatic primary tumor
is shown (light blue) on the left
side of all diagrams. Four
models are presented:
A, Metastasis is caused by rare
variant clones that develop in
the primary tumor;
B, Metastasis is caused by the
gene expression pattern of most
cells of the primary tumor,
referred to as a metastatic
signature;
C, A combination of A and B, in
which metastatic variants
appear in a tumor with a
metastatic gene signature;
D, Metastasis development is
greatly influenced by the tumor
stroma, which may regulate
angiogenesis, local invasiveness,
and resistance to immune
elimination, allowing cells of
the primary tumor, as in C, to
become metastatic.

38. Are there genes whose principal or sole contribution to
tumorigenesis is to control metastasis?
 genes that function as “metastasis oncogenes” or
“metastatic suppressors” are rare
 At least a dozen genes lost in metastatic lesions have been
confirmed to function as “metastasis suppressors”
 Their molecular functions are varied and not yet
completely clear; however, most appear to affect various
signaling pathways
 Recent work has suggested that two miRNAs, mir335 and
mir126, suppress the metastasis of breast cancer, while a
second set (mir10b) promotes metastasis

40. Epithelial cells
 1. Closely adjoined
 2. Polarized
Epithelial Markers:
 E-Cadherin (adherens junctions)
 Claudins (tight junctions)
 Occludin (tight junctions)
 Desmoplakin (desmosomes)
 Cytokeratin-8, -18 and -19
 Mucin-1
There are 5 different types of cell junctions. They are tight
junctions, adherens junctions, desmosomes, hemidesmosomes,
and gap junctions.

41. Mesenchymal cells
 1. Not adjoined
 2. No polarity
Mesenchymal Markers:
 Vimentin
 N-Cadherin
 Fibronectin
 Vitronectin
 FSP1(fibroblast-specific
protein 1)
 Smooth-muscle actin
 FGFR2 IIIb and IIIc splice
variants

42.  Epithelial cells can convert into mesenchymal cells by a
process known as EMT, which disrupts cell-cell adhesion and
cell-ECM adhesion.
 * Embryogenesis and development
 * Wound healing
 * Fibrosis
 *Cancer

43. EPITHELIAL PLASTICITY IS BI-DIRECTIONAL
 MESOTHELIOMA
 SYNOVIAL SARCOMA

44. CELLULAR MODIFICATIONS ASSOCIATED
WITH EMT
 1. In vitro morphology and function
* stellate or spindle shape
* resistance to anoikis
* increased migration
* invasion to collagen matrix
 2. Down regulated proteins
* E- Cadherin
* Cytokeratin
* Occludin
* Claudin

45.  3.Up regulated Proteins
* N – Cadherins
* vimentin SNAI1
* Snai2
* Twist
* MMPs (2,3,9)
* Integrins αy β6
 4.Activated proteins
* ILK
* GSK – 3 β
* Rho
 5. Nuclear expression of proteins
* β catenin
* Smad ( 2,3 )
* Snail1
* Snail2
* Twist

46. Genes
SNAI1
Smad
Zeb
TWIST

47. Pathways
1. Wnt PATHWAY
*REDUCED CELL ADHESION – DOWNREGULATION
OF E- CADHERIN
***SNAI1 - INTERMEDIATE SIGNALLING PATHWAY ***

49. 2.Rac , FAK , Paxillin
• INCREASED MIGRATION
** SNAI2 – INTERMEDIATE SIGNALLING END
POINT***

51. 3. RAS , MAPK
 * CYTOSKELETON ACTIVATION
 * MIGRATION
 * FOCAL ADHESION REARRANGEMENT
**SNAI2 – INTERMEDIATE SIGNALLING PATHWAY***

52. 3.

53. 4. RhoA
 * STRESS FIBRES MIGRATION

58. EMT and Cancer
 • Progression of most carcinomas is associated with the
acquisition of mesenchymal phenotype.
 • Cells with an EMT phenotype induced by different
factors are rich sources for cancer stem-like cells.
 • Moreover, induction of EMT in tumor cells not only
promotes invasion and metastasis but also contributes
to drug resistance

59. Induction of EMT Generates Stem-Like Cells
 Mani SA, et al. The Epithelial-Mesenchymal Transition
Generates Cells with Properties of Stem Cells.
 Leukemia – initiating cells are CD34+CD38- cells.
 Colon cancer – initiating cells are CD133+ cells.
 Brain cancer – initiating cells are CD133+ cells.
 Prostate cancer – initiating cells are CD44+α2β1+ cells.
Breast cancer – initiating cells are CD44+ CD24- cells.
│ │
EMT phenotype EMT induction

60. miRNAs Link EMT to Stem-Like Cells in Human Cancers
miR-200 family and ZEB1/2
 miR-200a
* Knockdown of Akt-1 decreases the expression of miR-200 family including
miR 200a, and increases mammosphere forming ability in breast cancer
 miR-200b
* miR-200b inhibits expression of ZEB1, ZEB2, Lin28B and Notch1 in prostate
cancer
* miR-200b targets Suz12 and contributes to cancer stem cells maintenance in
breast cancer
 miR-200c
* miR-200c inhibits expression of ZEB1, ZEB2 and Bmi1 in breast cancer;
* miR-200c inhibits expression of ZEB1, Sox2, Bmi1 and KLF4 in pancreatic
cancer
 miR-183
* ZEB1 represses miR-183 expression, which increases the expression of Bmi1 and
KLF4 in pancreatic cancer
 miR-203
* ZEB1 represses miR-203 expression, which increases the expression of Bmi1
and KLF4 in pancreatic cancer

61.  Several miRNAs have been identified as either oncogenes (miR-
17–92, miR-155, miR-21) or tumor suppressors (miR-15a, miR-16a,
let-7) and some human tumor types can be classified by miRNA
signatures.
• The miR-200 family of miRNAs consists of five members (miR-
200a,200b, miR-200c, 141, 429) that have been demonstrated to
have a role in EMT through regulation with the ZEB transcription
factors and regulation of E-cadherin and vimentin expression.
• The most striking effect of miR-200 expression was a change in
protein constituents in the media resulting from protein
secretion and shedding with downregulation of extracellular
matrix, peptidases and cell adhesion proteins.
• Proteins upregulated with miR-200 restoration were associated
primarily with cytoskeletal regulation and cell adhesion
- Cancer Research (2011) Dec 15; 71(24): 7670–7682

62. WOUND HEALING
 Mainly in skin and cornea
 Wounding of epidermis
 Epithelial stem cells are activated .
 The adherence junctions disassemble and E- Cadherin
expression is down regulated , vimentin expression is up
regulated
 cytoskeleton is activated
 Cell start to migrate .
*SNAI2 – PLAYS AN IMPORTANT ORCHESRATING ROLE IN
THIS PROCESS *

63. EMT and Fibrosis
 Fibrosis is characterized by the presence of an excess of
fibrous connective tissue in an organ, and in particular
by an excessive deposition of collagen I.
 Renal fibrosis, for example, has been associated with
the activation of interstitial fibroblasts, which give rise
to collagen secreting myofibroblasts. In addition,
myofibroblasts can also originate from renal tubular
epithelial and endothelial cells that undergo EMT.
 High Snail1 expression and evidence of EMT has also
been found in the kidneys of patients with renal
fibrosis (Boutet et al, 2006).

65. Cancer and Fibrosis are (Induced by) Inflammation
 In the context of a chronic inflammatory condition,
TGFβ1and hypoxia activate EMT that converges in the
activation of NFκB, which is also induced by the
inflammatory cytokines and oxidative stress.

67. Importance of EMT concept in the
diagnosis and treatment of cancer
1. Specific (ant)agonists for key molecules in the process of
invasion and metastasis – “ Targeted therapy “.
Eg: Cystatin C to inhibit EMT in breast cancer .
2. TWO problems - find solutions in the application of
new knowledge
* the borderline between non invasive and invasive
neoplasia
* prediction of tumor cell behaviour
-- IMMUNOHISTOCHEMISTRY – used to detect expression
of molecular markers of invasion.

68. References :
 1. Kumar,Abbas,Fausto . Robbins and Cotran
Pathologic basis of disease,9th ed. Elsevier .
 2. Recent advances in histopathology vol -22
 3. Kalluri R , Weinberg RA. The basics of epithelial
mesenchymal transition . The journal of clinical
investigation .2009;119(6):1420-28.
 4. Boutet A, De Frutos CA, Maxwel PH , Mayol et al .
Snai1 activation disrupts tissue homeostasis and
induces fibrosis in the adult kidney. The EMBO
journal . 2006;25(23):5603-5613.
 5.Internet sources .