Advanced tumor immunology prof dr.ihsan edan alsaimary university of basrah - college of medicine

1. Prepared by
Prof.dr. ihsan edan alsaimary
Department of microbiology – college of
medicine – university of basrah
ihsanalsaimary@gmail.com mobile:009647801410838

2. Tumor
 Cells that continue to replicate,
fail to differentiate into
specialized cells, and become
immortal.
1. Malignant: A tumor that
grows indefinitely and spreads
(metastasis)--also called
cancer: kills host
2. Benign: A tumor that is not
capable of metastasis: does not
kill host
muscle, nerve, bone,
blood

3. Development of Tumor Cells
Neoplasm is an abnormal mass of tissue, the growth of which
exceeds and in uncoordinated with that of normal body tissue.

4. Properties of Cancer Cells
Cancer cells
1) clonally derived
2) changes in in vivo and in vitro growth
3) altered tissue-specific affinities
4) biochemical changes
5) chromosomal abnormalities

5. T cell responses to tumors
5

6. Cross-presentation of tumor antigens
6

7. Coussens et al. Science 339:286, 2013
M2
Immune responses that promote tumor growth
7

8. Coussens et al. Science 339:286, 2013
M2
Immune responses that promote tumor growth 8

9. 1863 1898 1957 1983 1985 1991, 4 2002 2009 2010 2011 2014
Description of
immune
infiltrates in
tumors by
Virchow
Treatment of
cancer with
bacterial
products
(“Coley’s
toxin”)
Cancer
immuno-
surveillance
hypothesis
(Burnet,
Thomas)
1976
Treatmen
t of
bladder
cancer
with BCG
IL-2
therapy
for
cancer
Adoptive
cell
therapy
Discovery
of human
tumor
antigens
(Boon,
others)
Adoptive
T cell
therapy
HPV
vaccination
in VIN
FDA approval of
sipuleucel-T (DC
vaccine) in
prostate cancer
FDA approval of
anti-CTLA4
(ipilumimab) for
melanoma
FDA approval
of anti-PD1
for melanoma
The history of cancer immunotherapy: from empirical
approaches to rational, science-based therapies
Breakthrough
status for
CAR-T cells
in leukemia
9

10. Types of Cancer
 Carcinoma: arising from epithelial tissue, such as glands,
breast, skin, and linings of the urogenital, digestive, and
respiratory systems (89.3% of all cancers)
 Sarcoma: solid tumors of muscles, bone, and cartilage that
arise from the embryological mesoderm (1.9% of all cancers)
 Leukemia: disease of bone marrow causing excessive
production of leukocytes (3.4% of all cancers)
 Lymphoma, Myeloma: diseases of the lymph nodes and
spleen that cause excessive production of lymphocytes (5.4%
of cancers)

11. Tumor immunology
* Transformation of normal cells to malignant cells by:
a- Spontaneous mutation during daily cell division
chemical carcinogens
b- It may be induced by physical carcinogens
viruses
* Cells become antigenically different from normal cells
* They are recognized and destroyed by immune system

12. Etiology Of Tumor
1) Inherited :
Expression of inherited oncogene
e.g. viral gene incorporated into host gene
2) Viral:
- Human papilloma, herpes type 2, HBV, EBV (DNA)
- Human T-cell leuckemia virus (RNA)
3) Chemical:
- Poly cyclic hydrocarbons cause sarcomas
- Aromatic amines cause mammary carcinoma
- Alkyl nitroso amines cause hepatoma
4) Radiological: Ultraviolet & ionizing irradiation
5) Spontaneous: failure in the cellular growth control

13. Tumor Associated Antigens
!) Viral Antigen :
a- Viral proteins and glycoproteins
b- New antigens produced by virally infected host
cells under control of viral nucleic acid
2) Tumor specific antigens :
- Tumor cells develop new antigen specific to
their carcinogens
3) Tumor specific transplantation antigens :
- Tumor cells express new MHC antigens due to
alteration of normally present MHC antigens

14. Tumor Antigens
 TSTAs
• Tumor Specific Transplantation Antigen
• not on normal cells.
 TATAs
• Tumor Associated Transplantation Antigen
• present on t.c.+ some normal cells

15. TSTAs
 Unique to tumor cells
 DO NOT occur on normal cells in the body
 Novel proteins created my mutation
presented on class I MHC
 Can either be chemically/physically
induced or virally induced tumor antigens

16. Genes
for TSTAs

17. TATAs
 NOT unique to tumor cells
 DO occur on normal cells in the body
 So where’s the problem?
• Fetal/adult presence
• Concentration of Growth Factors and
Growth Factor Receptors

18. TATAs cont’d
 Oncofetal Tumor Antigens (AFP &
CEA)
• Normally appear in fetus before
immunocompetence
• Later recognized as non-self
 Oncogene Proteins
 Human Melanomas

19. Tumour Antigens
• Tumour specific Antigens
– chemically induced
– virally induced
• Tumour associated antigens
– oncofetal tumour antigens
– oncogene proteins

20. Tumor Specific Antigens
1) Mutated tumor cell proteins
2) Tumor-specific mutated oncogenes or
tumor suppressor genes
3) Over-expression of normal antigens
4) Viral oncogene expression on surface
of the cell

21. Tumor Associated Antigens
• Human Chorionic Gonadotropin (HCG)
• Alpha Fetoprotein (AFP)
• Prostate Specific Antigen (PSA)
• Mucin CA 125 (glycoprotein molecules on
both normal epithelium and carcinomas)
• Carcinoembryonic Antigen (CEA)

22. Tumor Associated Antigens
4) Oncofetal antigens:
a- Carcino-embryonic antigens (CEA)
- Normally expressed during fetal life on fetal gut
- Reappearance in adult life:
GIT, pancreas, biliary system and cancer breast
b- Alpha fetoprotein:
- Normally expressed in fetal life
- Reappearance in adult life; hepatoma

23. Onco-fetal antigens
 Onco-fetal antigens may appear due to de-
repression of genes that were only expressed
early in life.
 Two major onco-fetal antigens are
1- alpha-fetoprotein (AFP)
AFP is produced only as a secreted protein
2- carcino-embryonic antigen (CEA )
CEA is found both on cell membranes and in
secreted fluids.
 Since secreted antigens contribute little toward
immunity against tumors, the role of these neo-
antigens in immuno-surveillance is questionable

24. Alpha-fetoprotein
 The normal range of AFP
concentrations in humans is 0-20
ng/ml.
 This level rises considerably in
patients with hepatomas and non-
seminal testicular carcinoma.
 A 5-fold or higher rise in this protein
is used for monitoring hepatomas and
testicular cancers.
 AFP level may also be raised in some
non-malignant conditions, such as
cirrhosis, in hepatitis and other forms
of liver damage.

25. Carcinoembryonic antigens
 CEA levels in normal people range up to
2.5 ng/ml,
 They increase significantly in certain
malignancies, particularly colo-rectal
cancers.
 They may also rise in some non-malignant
conditions (such as chronic cirrhosis,
pulmonary emphysema and heavy
smoking).
 Levels that are 4-5 times normal have
been used to predict recurrence of colo-
rectal tumors.

26. Tumor Antigens
Tumor „Specific“ Ag
1) MHCI plus abnormal cell proteins (Brc-Alb, Philadelphia chromosome, CML)
2) MHCI plus viral proteins(EBV, SV40, polyoma virus)
3) Abnormal glycosylation
4) Idiotypes of myelomas and lymphomas
Tumor Associated Antigens
1) Oncofetal Ag (alphafetoprotein – hepatoma, carcinoembryonic Ag – colon ca.)
2) Melanoma Ag (MAGE-1, Melan-A)
3) Her/neu Ag (GFR)
4) EPCAM (epithelial cell adhesion molecule, carcinomas)
5) Differentiaion Ag (CALLA: common acute lymphoblastoid leukemia
antigen CD10 pre-B cells)

27. How does metastasis occur ?
 Three routes of spread
• Direct seeding of body cavities or
surfaces
• Lymphatic
• Heamatogenous

29. Primary Tumour
Basement Membrane
Extracellular Matrix
Basement Membrane
Adhesion to and Invasion
of Basement Membrane
Intravasation
Adherence to
Basement Membrane
and Extravasation
Metastasis
Haematogenous
spread

30. Molecular Changes
 Alteration in expression of adhesion
molecules
 Expression of proteolytic enzymes
 Expression of growth factors

31. Metastasis – Adhesion molecules
 Integrins
 Cadherins
 Selectins
 Immunoglobulins

32. Alteration in cell adhesion-Integrins
•Cell : ECM interactions
•Cell : Cell (heterotypic)
•Trans-membrane glycoprotien
•α and β subunit
•Over 20 members

33. Integrins-α and β subunit pairings

34. Changes in integrins in cancer
 Collagen receptors such as α2β1
down-regulated
 α4β1 and αvβ3 up-regulated in
melanoma

36. E-cadherin
 Epithelial cell adhesion molecule
 Down-regulated in many human
cancers
 Mutated in hereditary gastric cancer

37. Proteolytic Enzymes
 Serine proteases
• uPA, elastase, plasmin
 Matrix metalloproteinases
• Gelatinase, collagenase, stromolysin,
matrilysin
 Cysteine proteases
• Cathepsin B and L

38. Invasion - Altered expression of proteolytic enzymes
•Cancer cell intravasates into vessels (blood or lymphatic) by digesting the basement
membrane of the blood vessel with serine and matrix metalloproteases;
MMP’s (Zn2+-dependent) proteases e.g.
•then “migrates” in the blood stream
+ve
Cancer cell
Urokinase-
Plasminogen
activator
Plasminogen
Plasmin(S)
Collagen
Pro-MMP
Active MMP
Laminin
Fibronectin
TIMPs
-ve

39. Conclusion
 Metastasis is the cause of death in
most cancer patients
 Multi-step process
• Changes in adhesion, proteolysis and
growth factor expression
 Sites influenced by anatomy and
tropism
 The evolution of the metastatic
phenotype is uncertain

40. 1. The tumor is non-immunogenic at the beginning.
2. Weak response to tumors due to poor Ag-
presentation.
3. Loss of MHC-Ags leading to inability to present
tumor Ag peptides.
4. Presentation of MHC-Ag complexes to the TCR in
the absence of costimulation, this may lead to
anergy.
5. Tumor cells may also lack other molecules required
for adhesion of lymphocytes such as LFA-1, LFA-3 or
ICAM-1, or they may express molecules such as
mucins, which can be anti-adhesive.

41. 6. Tumor may also secrete immunosuppressive
cytokines such as TGFB and vascular endothelial
growth factor (VEGF), and prostaglandin E2 that
inhibit development and proliferation of T-cells.
7. Growth in immunologically privileged sites.
Recent evidence has shown that some tumors
express FasL, by which they induce apoptosis in
Tc cells expressing Fas (this is the direct
opposite of what the immune system intended to
happen).

42. Tumor Markers
* They are either Tumor antigens or Tumor products
1- Tumor Antigens
1) Alpha fetoprotein antigen (AFP) in cases of hepatoma
2) Carcinoembryoinic antigen (CEA) in gastrointestinal
tumors, tumors of biliary system and cancer breast
3) Cancer antigen 125 (CA 125) in ovarian carcinoma
4) Cancer antigen 15-3 (CA15-3) in breast cancer
5) Cancer antigen 19-9 in colon and pancreatic tumor
6) Prostatic specific antigen (PSA) in prostatic tumors

43. COMMON TUMOR MARKERS
Analyte Cancer Use
CEA Monitor colorectal, breast, lung cancer
CA-125 Ovarian cancer monitoring
CA15-3, 27. 29 Monitor recurrences of breast cancer
AFP Germ cell tumors, liver cancer
Total PSA Screen and monitor prostate cancer
Free PSA Distinguish prostate cancer from BPH
HCG Germ cell and trophoblastic tumors
Hormone
receptors Breast cancer therapy
NMP 22, BTA
FDP Monitor recurrences of bladder cancer

44. CEA
• Described by Gold and Freedman in1965 as a
marker for Colorectal Cancer
• Molecular mass of approximately 200,000 kA
• Glycoprotein with a carbohydrate composition
ranging from 50 - 85% of molecular mass
• CEA levels 5 - 10 times upper limit of normal
suggests colon cancer
• CEA is not used to screen for colon cancer

45. CA-125
• CA-125 glycoprotein molecular weight 200-1,000 kda
• Introduced in 1983 by Bast for ovarian cancer
• In the US, in 1998 25,400 new cases will be diagnosed
and 14,500 women will die as a result of this disease
• 70% of the women with ovarian cancer are over the age
of 50
• One half of patients with stage 1 ovarian cancer have
elevated CA-125 levels and a five year survival rate of
90%. In late stage disease, the five year survival rate is
from 4-30%
• Worldwide incidence is highest in industrialized
countries and lowest in Japan and India

46. SYMPTONS OF OVARIAN CANCER
• ASCITES
• ABDOMINAL and PELVIC PAIN
• ABNORMAL UTERINE BLEEDING
• GASTROINTESTINAL DISCOMFORT
• WEIGHT LOSS
• URINARY FREQUENCY

47. RISK FACTORS
INCREASED RISK
Family History
Advanced Age
Infertility
Nulliparity
DECREASED RISK
Oral Contraceptive
Breast Feeding
Tubal Ligation

48. GUIDELINES FOR ORDERING/
INTERPRETING TUMOR MARKER TESTS
• Never rely on the result of a single test
• Order every test from the same laboratory
• Consider half-life of the tumor when interpreting the
result
• Consider how the Tumor Marker is removed or
metabolized
• Consider Hook Effect
• Consider presence of HAMA antibodies

49. MULTIPLE MYELOMA
Multiple Myeloma - proliferation of a single clone of
plasma cells that produces a monoclonal protein
Annual Incidence - 4 in 100,000
Number of cases per year - 13,000
Represents 1% of all malignant diseases
Median age at diagnosis - 65 years
Median survival - 3 years

50. ETIOLOGY OF MULTIPLE MYELOMA
Radiation Exposure
Agriculture - Farming & Pesticide Use
Chemicals - Benzene
Not Related to Smoking or Alcohol Consumption

51. Incidence of breast
cancer :
• Rare before age 20, rises steadily
around the age of menopause
• In USA and Canada 1 in 8 women will
develop breast cancer.
• In Indonesia , second cause of
mortality in women after cervix
cancer

52. Risk factors
• Maternal relative with breast cancer
• BRCA1 and BRCA2 genes.
• Longer reproductive span.
• Obesity
• Nulliparity
• Later age at first pregnancy.
• Atypical epithelial hyperplasia
• Previous breast ca.

53. Can Breast Cancer Be
Found Early ?
• In USA screening mammogram
every year for women aged 40 and
older.
• Between aged 20-39 , clinical breast
examination by a health professional
every 3 years.
• Women aged 20 or older BSE every
month

54. How Is Breast Cancer
Diagnosed?
• Signs and Symptoms
• Medical History and Physical
Examination
• Imaging Test for Breast Disease
Diagnosis
• Nipple Discharge Examination
• Biopsy

55. IHC for Breast Cancer
• Steroid Hormone Receptors
• HER-2/ neu / c-erbB-2
• p53
• bcl-2
• nm23H1
• Ki-67 /MIB
• MUC1

56. Steroid Hormone Receptors
Estrogen Receptor/Progesteron Receptor
ER/PR
• Predict the response rate to
endocrine therapy
• Positive hormone receptor status is
a favorable prognosis.

57. Oncogene : c-ErbB-
2/neu/HER-2
• neu/HER-2 oncogene encodes a 185
kDa transmembrane glycoprotein.
• The neu/HER-2 gene product
possesses tyrosine kinase activity.
• Overexpression in about
30% Ca. Related to resistance
chemoth/ , grow and spread more
agressively
• Treated with HERCEPTIN

58. IHC of C-ErbB-2/neu/HER-2:

59. p53
• P53 is tumor suppressor gene which
can induce apoptosis after
irreparable cell damage and regulates
the normal cell growth cycles by
activating the transcription of
involved gene.
• Inactivation or mutation of p53 leads
to replication of damaged DNA thus
promoting the development of
malignant cell clones.

60. • Overexpression of mutant p53 can
be detected by IHC.
• Overexpression of p53 seems to be
related to resistance against certain
regimens of endocrine and cytostatic
chemotherapy.

61. IHC of p53

62. Bcl-2
• bcl-2 is a suppressor of apoptotic
cell death.
• The expression of bcl-2 is
correlated positively with higher
estrogen receptor content.

63. IHC of bcl-2

64. Ki-67 / MIB-1
• Ki-67 is nuclear protein which is
expressed in the S,G2 and M phase of
dividing cells.
• Expression of Ki-67 is used as an
index of proliferative activity in
breast cancer.
• The presence of <10% low rate,
10-20% intermediate and >20% is
• high proliferative rate.
•

65. IHC of Ki-67

66. Conclusions :
• Immunohistochemistry is a staining
technique for detecting many
prognostic factors of breast cancer.
• The accurate examination and
reporting of standard pathologic
features remains very important in
the diagnosis and prognosis of women
with breast cancer.

67. 2- Tumor Products
a) Hormones :
- Human chorionic gonadotrophins (HCG) are secreted
in cases of choriocarcinoma
- Thyroxin (T3 & T4) is secreted in cases of cancer
of thyroid gland
b) Enzymes :
- Acid phosphatase enzymes in cases of cancer prostae
- Alkaline phosphatese, lipase and amylase enzymes in
cases of cancer pancreas

68. Immune Surveillance System

69.  The immune system continually surveyed the body
for the presence of abnormal cells, which were
destroyed when recognized.
 immune surveillance:
Immune response normally eliminates malignant cells
regularly; thereby preventing the establishment
and growth of tumors.
If there is immune disorder the tumor will established
and grow.
 This may be evident by:
• Many tumors contain lymphoid infiltrates.
• Spontaneous regression of tumors occurs.
• Tumors occur more frequently in the neonatal and in old
age, when the immune system function less effectively.
• Tumors arise frequently in immunosuppressed
individuals.

70. Immune Surveillance System
This system include :
1) Natural killer (NK) cells
They kill directly tumor cells,helped by interferon, IL-2
2) Cytotoxic T-cells
They also kill directly tumor cells
3) Cell mediated T-cells (effector T-cells)
They produce and release a variety of lymphokines :
a-Macrophage activation factor that activate macrophag
b-Gamma interferon and interleukin-2 that activate NK
c-Tumor necrosis factor (cachectine)

71. Immune Surveillance System
4) B-cells :
- Tumor associated antigens stimulate production of
specific antibodies by host B-cells
- These specific antibodies bind together on tumor cell surface
leading to destruction of tumor through:
a- Antibody mediated-cytotoxicity :
kill
Cytotoxic T-cells IgG-coated tumor cells
b- Activation of macrophages
release
Sensitized T-cells macrophage activating factor
IgG-coated tumor cells macrophages activate
c- Activation of classical pathway of complement leading to
Lysis of tumor cells

72. Tumor Escape
Mechanisms by which tumor escape immune defenses:
1) Reduced levels or absence of MHCI molecule on
tumor so that they can not be recognized by CTLs
2) Some tumors stop expressing the antigens
These tumors are called “antigen loss variants”
3) Production of immunosuppressive factors by tumor
e.g. transforming growth factor (TGF-β)
4) Tumor antigens may induce specific immunologic
tolerance

73. Tumor Escape
5) Tumor cells have an inherent defect in antigen
processing and presentation
6) Blocking of receptors on T-cells by specific antigen
antibodies complex (after shedding of tumor Ag)
prevents them from recognizing and attacking
tumor cells
7) Antigens on the surface of tumors may be masked
by sialic acid-containing mucopolysaccharides
8) Immune suppression of the host as in transplant
patients who show a higher incidence of malignancy

74. Four
mechanisms of
oncogene
activity to
deregulate cell
division

75. Failure of Immune Response Against Tumor
An effective tumor response to tumor can be hampered by the
following factors: if effector cells exist and tumors are
antigenic,why aren’t tumors rejected?)
a. Site of tumor. Some tumors arise in areas not accessible to
effector cells (eye and central nervous system).
b. Antigenic modulation. Tumor cells may undergo several
antigenic changes.
c. Blocking factors. Immune complexes and cytophilic antibodies
can mask surface tumor antigens or prevent binding by effector
cells or lytic antibodies.

76. Immuno-Diagnosis
 Monoclonal antibodies labeled
with radioisotope have been used for
in vivo detection of relatively small
tumor foci.
 Antibodies have also been used in
vitro to identify the cell origin of
undifferentiated tumors, particularly
of lymphocytic origin.
 Immuno-histological staining is
used to confirm suspected metastatic
foci, especially in bone marrow.

77. Immunotherapy
 Immunotherapy has been used as
adjunct to traditional treatments.
 Both active and passive means of
stimulating the non-specific and
specific immune systems have been
employed, in some cases with
significant success

78.  A variety of immunopotentiating agents
(biological response modifiers) are used to
enhance anti-tumor immunity. They include:
 bacterial products
 synthetic chemicals
 cytokines
 Most of these agents exert their effects by
activating macrophages and natural killer (NK)
cells, eliciting cytokines or enhancing T-cell
functions.
.

79. A number of cytokines have been used to potentiate
the immune function of the host
Anti-tumor
mechanism(s)
Tumor types
Cytokine
Increased expression
of class I MHC,
possible cytostatic
anti-tumor effect
Remission of hairy cell
leukemia,
IFN-alpha, beta
Increased MHC
antigens; macrophage,
Tc and NK cell
activation
Carcinoma of ovary
IFN-gamma
T cell proliferation and
activation of NK cells
Renal carcinoma and
melanoma
IL-2
Macrophages and
lymphocyte activation
Reduce malignant
ascites
TNF-alpha

80. CANCER
VACCINES
Antigen vaccines
These vaccines are made from special proteins
(antigens) in cancer cells. Scientists have worked out
the genetic codes of many cancer cell proteins
Whole cell vaccines
A whole cell vaccine uses the whole cancer cell, not
just a specific cell protein (antigen), to make the
vaccine

81. Dendritic cell vaccines
Dendritic cells help the immune system recognise
and attack abnormal cells, such as cancer cells.
DNA vaccines
These vaccines are made with DNA from cancer
cells.

84. Short Answer Questions

85. • Explain how some cancer cells that can
make TGF-beta are immunosuppressive.
• Tumours and transplants are similar to one
another,yet very different. Explain this
observation in the context of what the
immune system recognizes and the result of
this recognition.
• The qualities of proliferation and
differentiation are essentially all that
distinguishes a normal cell from a cancer
cell. Explain.

86. • Design an experiment using mice that proves
that the immune system provides immunity
against tumours.
• Distinguish between tumour-specific
transplantation antigens (TSTA) and tumour
associated transplantation antigens (TATA).
• Design an experiment to show Tumour
associated Transplantation Antigens (TATA).
• What is the main difference separating cell
surface antigens from chemically induced and
virually induced cancers?

87. • Speculate on why this difference leads to
difficulty in designing anticancer vaccines.
• What are oncofetal antigens? Are they
important in tumour immunity? Why?
• What is immune surveillance?
• All evidence for immune surveillance is
indirect. Speculate on how you could get
direct evidence.

88. • What immune cells play a role in tumour
rejection? Briefly describe how each
accomplishes this task. Include such things as
cytokines, perforins, ADCC etc.
• Cancers camouflage themselves to evade
antitumour defenses. Pick three possible
forms of camouflage that you think are most
important, describe them and state why you
think they are most important.
• What are immunotoxins?

89. • Surgery, radiation and chemotherapy are
the methods most widely used to treat
cancer patients. What are the problems
with this regimen, and how could
immunotherapy overcome these problems.
• Distinguish between specific and
nonspecific immunotherapy.