DR. Mohamed Labib Salem, PhD Prof. of Immunology Faculty of Science, Tanta University, Egypt

1. Dr. Mohamed Labib Salem, PhD
Prof. of Immunology, Faculty of Science
Director, Center of Excellence in Cancer Research
TANTA UNIVERSITY
Cellular and Molecular
Tumor Markers
February 7, 2019
Luxor, EGYPT

3. Lecture Contents
Tumor and Cancer
Types of tumor markers
Applications of tumor markers
Evaluation of tumor markers
Measuring tumor markers
Cellular tumor markers
Production of tumor markers

4. What Is Cancer?
• "Cancer" is a group of more than one
hundred separate diseases characterized
by an abnormal and unregulated cell
growth.
• This growth destroys surrounding body
tissues and may spread to other parts of
the body in a process that is known as
metastasis

5. What Is Cancer?
• Carcinomas: arise from epithelial tissues. E.g.
Lung, breast, and colon.
• Sarcomas: arise from connective tissues.
• Lymphomas: arise in the lymph nodes and
tissues of the body's immune system.
• Leukemias: arise from the immature blood
cells that grow in the bone marrow and tend
to accumulate in large numbers in the
bloodstream

8. Tumor versus Cancer
Invasion versus Metastasis

10. Intravasation/extravasion
Invasion
Osteoclasts
Osteoblasts
Fibroblasts
Hematopoeitic Progeintors
Kupffer cells
Gilial cells
Endothelial cells
Immune cells
Angiogenesis
Dormancy
Dormancy
Metastatic colonization
What Is Cancer?

12. Models of Tumor Metastasis
Klein, Science 321, September, 2008

16. Tumor Markers
Types and Applications

17. Tumor
Markers
Cellular
Markers
1
2
3
14
5

18. What are Tumor Markers?
• Are substances produced by cancer cells,
or produced in response to the cancer's
presence.
• Are found in blood, other body fluids.
• Are substances identifiable by either
mAbs, polyclonal Abs or both.

19. What are Tumor Markers (TMs)?
• Most are proteins.
• Some TMs are associated with only one
type of cancer.
• Others TMs are associated with two or
more cancer types.
• There is no “universal” TM that can
detect any type of cancer.

20. History of Tumor Markers
• 1846 Bence-Jones Protein
• 1940 Acid Phosphatase
• 1960 Alpha-fetoprotein
• 1965 Carcinoembryonic antigen
• 1970 Oncogenes
• 1975 Monoclonal antibodies
• 1980 CA 125, PSA, Carbohydrate antigens
• 1980 Tumor suppressor genes
• 2001 Microarray, Mass spectrometry,
multi-parameters analysis
• 2005 circulating tumor cells

21. TYPES OF TUMOUR MARKERS
• Tumour-Associated Proteins (TAP)
• Cell membrane receptors
• Hormones
• Immunoglobulins / Cellular antigens
• Protein clusters and fragments
• Polyamines
• Chromosomal material
• Genes (single, clusters)
• Genetic material (DNA, RNA, mRNA)

22. WHO Criteria (1968)
1. Important role in evaluation
2. Role must be well understood
3. Role must be recognized
4. Can be tested early
5. Detects treatment response
6. Support for test available
7. Support / treatment beneficial
8. Benefits greater than side-effects
9. Screening must be cost-effective
10.Detect / diagnose malignant disease

23. Characteristics of an ideal TMs

24. Molecular Basis of TMs

25. Applications of Tumor Markers
• Detection
• Diagnosis
• Prognosis
• Treatment management (response to therapy)
• Post-treatment management (Recurrence)

26. Detection of elevated TM levels
0
15
30
45
60
Early Late
Elevated level
Normal level
Standard reference level

28. Specific tumor markers
1. Alpha-fetoprotein (AFP)
2. Beta-2-microglobulin (B2M)
3. Human chorionic gonadotropin (HCG)
4. Bladder tumor antigen (BTA)
5. Neuron-specific enolase (NSE)
6. Epidermal growth factor receptor
(EGFR/HER1)
1. HER2/neu, erbB-2, or EGFR2)
2. K-RAS (mutation in EGFR)
3. Hormone receptors (ER and PR)
4. Lactate dehydrogenase (LDH)
5. Prostate-specific antigen (PSA)
6. Prostatic acid phosphatase (PAP)
7. Prostate-specific membrane antigen
(PSMA)
15. Bcr-abl
16. CA 15-3; CA 27.29
17. CA 125; CA 72-4; CA 19-9
18. Bladder tumor antigen (BTA)
19. Calcitonin
20. Carcinoembryonic antigen (CEA)
21. Chromogranin A (CgA)
22. K-RAS (mutation in EGFR)
23. Immunoglobulins
24. Free light chains
25. Lactate dehydrogenase (LDH)
26. Prostate-specific antigen (PSA)
27. S-100 and TA-90 (melanoma)
28. Thyroglobulin

29. TMs currently used
with cancer types

30. CA15-3/CA27.29
• Cancer type: Breast cancer
• Tissue : Blood
CA19-9
• Cancer types: Pancreatic cancer, gallbladder
cancer, bile duct cancer, and gastric cancer
• Tissue : Blood
CA-125
• Cancer type: Ovarian cancer
• Tissue analyzed: Blood
To assess prognosis and if treatment is working

31. Carcinoembryonic antigen (CEA)
• Cancer : Colorectal cancer and breast cancer
• Tissue : Blood
Alpha-fetoprotein (AFP)
• Cancer : Liver cancer and germ cell tumors
• Tissue : Blood
Anaplastic lymphoma kinase (ALK) gene rearrangements
• Cancer : Non-small cell lung cancer and anaplastic large
cell lymphoma
• Tissue : Tumor
To assess prognosis and if treatment is working

32. Beta-2-microglobulin (B2M)
• Cancer : Multiple myeloma, chronic lymphocytic
leukemia, and some lymphomas
• Tissue : Blood, urine, or cerebrospinal fluid
Beta-human chorionic gonadotropin (Beta-hCG)
• Cancer : Choriocarcinoma and testicular cancer
• Tissue analyzed: Urine or blood
Calcitonin
• Cancer : Medullary thyroid cancer
• Tissue : Blood
To assess prognosis and if treatment is working

33. Prostate-specific antigen
(PSA)
• Cancer: Prostate cancer
• Tissue: Blood
Thyroglobulin
• Cancer: Thyroid cancer
• Tissue: Tumor
Immunoglobulins
• Cancer: Multiple myeloma
• Tissue: Blood and urine
To assess response to treatment/recurrence
21-Gene signature
(Oncotype DX)
• Cancer: Breast
cancer
• Tissue: Tumor
70-Gene signature
(Mammaprint)
• Cancer: Breast
cancer
• Tissue: Tumor

34. To monitor tumor recurrence
Chromogranin A (CgA)
• Cancer : Neuroendocrine
tumors
• Tissue : Blood
Chromosomes 3, 7, 17, and
9p21
• Cancer : Bladder cancer
• Tissue : Urine
Cytokeratin fragments
21-1
• Cancer : Lung cancer
• Tissue : Blood
HE4
• Cancer : Ovarian
cancer
• Tissue : Blood

35. BRAF mutation V600E
• Cancer : Cutaneous melanoma and colorectal cancer
• Tissue : Tumor
CD20
• Cancer : Non-Hodgkin lymphoma
• Tissue : Blood
KRAS mutation analysis
• Cancer : Colorectal cancer and non-small cell lung cancer
• Tissue : Tumor
To predict response to targeted therapies

36. Estrogen receptor (ER)/progesterone receptor (PR)
• Cancer: Breast cancer
• Tissue: Tumor
EGFR mutation analysis
• Cancer: Non-small cell lung cancer
• Tissue: Tumor
HER2/neu
• Cancer : Breast cancer, gastric cancer, and
esophageal cancer
• Tissue: Tumor
To predict response to targeted therapies

39. EVALUATION
TUMOR MARKERS

40. Tumor
Markers:
Qualities
• Ease of performance
• Low cost
• Patient acceptance
• Statistical factors such as:
(1)sensitivity,
(2)specificity,
(3)positive predictive value
(4)negative predictive value

41. Evaluation of a Tumor Marker Assays:
Sensitivity of the test
• The ability of the test to detect those
individuals with cancer in the test population.
• The True Positive (TP) ratio, that is, the
proportion of positive test results in all
individuals with disease.
• It is defined as the number of true positive
(TP) cases divided by the total number of
cancer cases
Sensitivity= TP/TP+FN

42. Evaluation of a Tumor Marker Assay:
Specificity of the tets
• Is defined as the ability of the test to identify
those free from cancer in the test population.
• The specificity of the test is the True Negative
(TN) ratio, that is, the proportion of negative
tests in all individuals without disease.
• It is defined as the number of TN cases
divided by the number of people without the
disease.
Specificity= TN/TN+FP

43. Evaluation of a Tumor Marker Assay:
Predictive Values
• The positive predictive value: (TP/TP+FP)
The measure of the validity of a positive test,
or in other words, the proportion of positive
tests that are true positive cases.
• The negative predictive value: (TN/TN+FN)
The measure of the validity of a negative test,
or, the proportion of negative tests that are
true negative cases.

44. Quality Requirements for the Use of TMs
• Pre-analytical requirements:
choice of tumor marker, specimen type,
specimen timing, sample handling.
• Analytical requirements:
assay standardization, internal and
external quality control, interferences.
• Post-analytical requirements:
reference intervals, interpretation, and
reporting of tumor marker results.

45. Some Helpful Tips for ordering TMs
1. Never rely on the results of a single test.
2. When ordering serial testing, be certain to
order every test for the same lab. Using
the same assay kit.
3. Be certain that the TM selected for
monitoring recurrence was elevated in the
patient prior surgery.
4. Consider the half-life of the TM when
interpreting test results.

46. Some Helpful Tips for ordering TMs
5. Consider how the TM is removed from
or metabolized in the blood circulation.
6. Consider ordering multiple TMs to
improve both the sensitivity and
specificity for diagnosis.
7. Be aware of the presence of ectopic
TMs.

47. • A small amount of TMs exist in normal
individuals.
• Some noncancerous conditions can cause the
levels of certain TMs to increase.
• Some people with cancer never have higher
levels of TMs.
• Some people with large cancer have low levels
of TMs.
• TMs have not been identified for every type of
cancer.
limitations to The Use of Tumor Markers

49. Methods
to
Measure
Tumor
Markers

50. Methods
to
Measure
Tumor
Markers

51. Methods to Measure Tumor Markers
• ELISA for proteins.
• PCR for genes
• Immunohistochemsitry for proteins
• Mass Spectrometry for proteins.
• Proteomic array
• Genomic analysis

52. SANDWITCH ELISA
Tumor markers: Common Methods

53. Phases of indirect ELISA

56. Phases of sandwich ELISA
1
2
3
4
5

58. Useful Links
• http://www.aacc.org/Search/Pages/results.aspx/
Results.aspx?k=tumor%20markers# (Tumor
Markers Quality Requirements)
• http://fdi.com/tm_pres/presentation/index.html
(Basics of Tumor markers from FUJIREBIO,
Diagnostics, Inc.)
• http://www.authorstream.com/Presentation/doc
pawan-744922-tumor-markers/
• http://www.cancer.gov/cancertopics/factsheet/D
etection/tumor-markers

59. CIRCULATING TUMOR CELLS
(CTC)
Cellular Tumor markers

60. CTCs
• 1869 Australian Medical Journal: A Case of
cancer in which cells similar to those in the
tumors were seen in the blood after death
1869;14:146.
• 1955 Acta Chiurgica Scandinavia: Cancer Cells
circulating in the blood: a clinical study on the
occurrence of cancer cells in the peripheral blood
and in venous blood draining the tumor area at
operation 1955;201:1.
• 1976 American Journal of Medicine:
Carcinocythemia: An acute leukemia-like picture
due to metastatic carcinoma cells 1976;60:273.

62. Flow Cytometer

64. Isolation of CTC from Peripheral Blood
• Two Key Issues:
– Enrichment of epithelial/tumor cells from RBC &
WBC
– Characterization to distinguish
• Tumor cells from blood components
• Tumor cells from normal cells

65. Labeling of CTC and Blood Cells
Circulating Tumor Cell
CK
Anti-
CK-PE
EpCAM
Leukocyte
Nucleus
DAPI
CD45
Anti -
CD45-APC
HER2+*
Circulating Tumor Cell
CK
EpCAM
Anti-EpCAM
Ferrofluid
Y
HER2
Anti-
HER2-
PE-Cy7
Nucleus
DAPI
Nucleus
DAPI
Anti-EpCAM
Ferrofluid
Anti-
CK-PE

66. Magnetic Cell Presentation
Trajectory of magnetically
labeled objects
Analysis
Cartridge

67. Analysis of Enriched CTC
• Semi-automated
fluorescence microscope
• Automatically scans a
complete reaction cartridge
in about 10 minutes
• Software algorithm
identifies CTC candidates
• Cell images are presented
in a gallery format for
confirmation as CTC by a
technician or pathologist
CellSpotter Analyzer

68. Analysis of Enriched CTC
Nucleus
Cytokeratin
Control
Leukocyte
Composite
Intact Tumor Cells

69. Correlation of CTC count with ovorall Survival

70. Correlation of CTC count with tumor
marker level

71. Correlation of CTC count with tumor
progression
Cancer type Cancer stage

72. POTENTIAL CELLULAR MARKERS
Tumor Markers
Myeloid-derived suppressor
(MDSCs) cells
Regulatory T (Treg) cells

73. Correlation of MDSC numbers with cancer
progression

74. Adeno Mela Brest
Stage IV

75. Increases
in
MDSC
numbers
after
chemotherapy

76. ddAC, but not ddT, was associated with significant increases
in circulating MDSC in early stage breast cancer patients

77. PRODUCTION OF ANTIBODIES
Tumor Markers

78. Hybridoma
Technology

79. Hybridoma
Technology

80. Summary
• Cancer is a multiple diseases
• Cancer detection by molecules and cells
• Most of TM are proteins
• No single tumor marker is available
• New tumor markers are needed
• New antibodies are needed
• ELISA is the most common methods
• Flow cytometry for cellular markers

81. Center of Excellence in Cancer
Research

82. Center of Excellence in
Cancer Research

83. IsoFlux™ System
The next generation of
CTC analysis is here
Fluxion Biosciences
© 2014 Fluxion Biosciences. All rights reserved.
Cell Purification Unit at CECR, Tanta
University

84. Our Flow Cytomter at CECR, Tanta
University

85. Our Genomic Unit at CECR, Tanta
University

86. Our Genomic Unit at CECR, Tanta
University

87. Proteomic Unit
at CECR, Tanta University

89. THANAK
YOU
ALL