Diagnostic tools in oncology

1. Diagnostics tools in Oncology
Mohammed Fathy Bayomy, MSc, MD
Lecturer
Clinical Oncology & Nuclear Medicine
Faculty of Medicine
Zagazig University

2. * Tumor markers.
* Immunohistochemistry (IHC).
* Immunocytology (Flow-cytometry).
* Cytogenetics (Classic, FISH, CIH, SIH).
* Molecular diagnosis (PCR, blots).
* Gene Expression Analysis (Microarray).
* HLA typing.

3. Tumor Markers

4. Definition
Cellular products of malignant tumor tissue, which can
be detected in peripheral blood and other body fluids,
i.e. soluble antigens, hormones, or enzymes which are
produced by solid tumors and can be detected in
circulating blood.

5. Classificatio
n
1- Tumor products
• Tumor-associated antigens: AFP, CEA, CA 19-9, CA 15-3, CA 125.
• Hormones: gastrin, calcitonin, insulin, ?HCG, catecholamines, VIP.
• Enzymes: NSE, PSA, LDH.
• Serum proteins: immunoglobulins, Bence Jones protein, thyroglobulin.
2- Tumor induced markers
• Acute phase proteins: ferritin, haptoglobin, B2-microglobulin, A2-
globulin.
• Enzymes: AP, GGT, LDH, GOT, GPT, CK.

6. Factors affect serum
level
1- Tumor parameters:-
• Tumor weight.
• Metabolic activity.
• Marker release due to active secretion /necrosis / apoptosis.
• Tumor perfusion / vascularization.
2- Therapy
Effective treatment may cause transient marker release due to oncolysis
(DD: tumor progression under therapy), followed by marker level
decrease /normalization

7. 3- Metabolic parameters:-
• Renal insufficiency.
• Liver insufficiency.
• Cholestasis.
4- Test method
Tumor marker test results can vary depending on the method applied
>>> inconsistent reference values with different tests (esp. with CEA
and CA 19-9), limited reproducibility with different methods

8. Indications
Tumor marker analysis tends to be of limited clinical consequence due to low
sensitivity and specificity, specific indications for their use are therefore
required:-
• Unsuitable for screening of asymptomatic patients (except PSA +
rectal digital examination and sonography in men over 50 years of
age).
• Unsuitable for primary tumor diagnosis.
• Unsuitable for proving malignancy in organ abnormalities (except B-
HCG detection or high AFP levels in men).

9. • Potentially suitable for assessment of risk groups or symptomatic
patients.
• Suitable in individual cases for prognostic evaluation
* CEA in colorectal carcinoma.
* AFP and B-HCG in germ cell tumors.
* B2-microglobulin in multiple myeloma.

10. Tumor marker analysis is mainly used for treatment evaluation and follow-up
of treated patients for better or earlier determination of tumor response
(relapse, metastasis).
• Increase in tumor markers may be detectable several months before
occurrence of clinical symptoms
• Clinically relevant conclusions may be derived from tumor marker kinetics,
not individual values

11. Whether tumor marker analysis is indicated depends on the clinical
relevance:-
• AFP and BHCG in germ cell tumors are meaningful due to therapeutic
relevance.
• CA 19-9 in metastatic pancreatic carcinoma is of limited value in palliative
situations, the approach is determined primarily by clinical symptoms.
Here, tumor marker analysis is not required.

12. Recommended Time Points for Tumor Marker Analysis
• Preoperatively
• Postoperatively: 2–10 days after surgery, then every 3 months, from third year
on: every 6 months
• Before changing treatment.
• Clinically suspected relapse or metastasis.
• Before continuing treatment of tumors that cannot be measured with
imaging techniques
• Restaging.
• 14–30 days after first detection of increased tumor marker levels.

13. Tumor markers

16. Immunohistochemistry

17. Definition
Use of tumor antigen (protein) mainly to
determine its differentiation (cell typing)
>>>> diagnostic role.
+
>>>> predictive role (predict response to treatment).
>>>> prognostic role (predict which patients will survive).

18. Tumor antigen
Include
1- Nuclear antigen.
2- Cytoplasmic antigen.
3- Membranous antigen.
4- Stromal antigen.

19. 1-Nuclear antigen
Include
1- Cycle cycle control proteins e.g: cyclins &
cyclin dependent kinases (CDK).
2- DNA replication enzymes e.g. Tdt, viral
proteins like polymerases and RNA reverse-
transcriptase.

20. 2-Cytoplasmic antigen
Include
1- Microfilaments e.g: actin.
2- Intermediate filaments e.g. cytokeratin (CK),
vimentin, desmin, neurofilament (NF), Glial
Fibrillary Acid Protein (GFAP).

21. 3-Membraneous (surface) antigen
Include
1-Membrane bound cell receptors e.g:
Her2/neu.
2-Adhesion molecules e.g. E-cadherin.

22. 4-Stromal antigen
Include
1- Extracellular matrix protein e.g: collagen,
laminin, fibronectin.
2- Abnormal deposits e.g. amyloid.

23. Immunoreactivity (Immunostaining)
Primary antibody react (bind) to its specific antigen
Secondary antibody link primary antibody to enzyme
system e.g. peroxidase to amplify reaction to be
easily detectable

24. Location of immunostaining
1-Membranous.
2-Cytoplasmic.
3-Nuclear.
4-Stromal.

25. Nuclear Cytoplasmic Membranous
* Beta-catenin
* ER
* PR
* Bcl-6
* Ki-67
* Cyclin D-1
* p63
* p53
* TdT
* TF-1
* WT-1
* Myogenin
* Telomerase
* CK
* Vimentin
* Desmin
* Actin
* CEA
* PSA
* HCG
* Thyroglobulin
* EMA
* Chromogranin
* Calretinin
* Osteonectin
* Synaptophysin
* Hep-par-1
* HMB-45
* Factor VIII
* Inhibin
* Calcitonin
* Melan-A
* nm23
* ALK
* Bcl-2
* CD68
* Beta-catenin
* CD1a
* CD99
* CD117
* CD44
* CD10
* Her2 (neu)
* CD20
* CD30
* CD11
* CD3
* Uroplakin

26. Extent of immunostaining
1-Negative.
2-Focal.
3-Patchy.
4-Diffuse.

27. Intensity of immunostaining
1-Negative.
2-Weak positive (+ or +1).
3-Moderate positive (++ or +2).
4-Strong positive (+++ or +3).

28. Clinical application
(I) Diagnosis of malignancy (diagnostic).
(II) Prediction of response to therapy (predictive).
(III) Prediction of prognosis (prognostic).

29. (I) Diagnostic markers
Include
1-Routine phenotyping of tumors.
2-Malignant tumor vs tumor like reactivity
Follicular lymphoma Reactive follicular hyperplasia
bcl2
Positive Negative

30. 3-Malignant tumor vs Benign tumor
Dermatofibrosarcoma Dermatofibroma
CD34
Positive Negative
4-Malignant tumors vs similar Malignant tumor
Mesothelioma Adenocarcinoma of lung
Calretinin Positive Negative
CEA Negative Positive

31. 4-Malignant tumors vs similar Malignant tumor
Adrenal cortical carcinoma Pheochromocytoma
Vimentin Positive Negative
Chromogranin Negative Positive
5-Phenotyping of problematic cases:-
* Undifferentiated malignant tumors.
* Metastasis of unknown origin.

32. ( )

33. (II) Predictive markers
Include
1- Hormone receptors (HR):- ER & PR in breast
cancer predict response to anti-hormonal
treatment e.g. Tamoxifen ‘’Nolvadex’’, AI
(Anastrazole ’’Arimidex’’) , AR in prostate cancer.

34. 2- HER2 receptor overexpression: in breast
cancer predict response to monoclonal
antibodies ‘’MoAb’’ e.g. Trastuzumab
‘’Herceptin’’.

35. 3- C-Kit (CD117): in GIST predict response to
Tyrosine Kinase Inhibitors ‘’TKI’’ e.g. Imatinib
‘’Gleevec’’.
4- Multidrug resistance gene (MDR-1)
expression: predict resistance to anthracyclins
& vinca alkaloids >>> shift to alternative
chemotherapeutic drugs

36. (III) Prognostic markers
Include
1- Proliferation markers:-
* Ki-67 (MIB-1) index: low (<2%)= benign & good prognosis,
intermediate (2-20%), high (>20%) = malignant & poor
prognosis.
* Proliferation cell nuclear antigen (PCNA).
* Bromodeoxuridine (Brd Urd).

37. 2- Invasiveness (local invasion) markers:-
* Loss of cell adhesion molecule expression e.g. E-Cadherin.
* Expression of matrix degrading enzymes (matrix
metalloproteinases eg. Type IV collagenase, cathepsin D

38. 3- Metastatic markers:-
* Endothelial marker (CD34) to measure microvessel density.
* Failure to express metastases suppressor gene (nm-23).
* Vascular Endothelial growth Factor (VEGF) expression
Quantitating as indictor for ability of new blood vessel
formation.
* Increase expression of endothelial adhesion molecule
(CD44)
* Cytokeratin staining in lymph node or bone marrow =
micrometastasis.

39. 4- Cancer genes alterations:-
* Overexpression of wild oncogenes: HER2 in breast cancer.
* Expression of oncogenes that normally not expressed: N-
myc in neuroblastoma, RET in thyroid cancer.
* Expression of mutated oncogenes: mutated K-RAS or N-
RAS in colon, mutated B-RAF in melanoma, mutated
EGFR or ALK in lung adenocarcinoma.
* Loss of expression of tumor suppressor genes: p53, Rb,
PTEN.

40. Carcinoma Markers
1- General markers:-
* Cytokeratin (CK).
* CEA.
* Hormone receptors.
2- Specific carcinoma markers.

41. Cytokeratin (CK)
Definition
Most complex member of intermediate filament that
present in epithelial cells linage.
Include
More than 20 protein, each given a number from 1
to 20 e.g. cytokeratin 7 ‘’CK 7’’.

42. Biological groups
1- High molecular weight-CK (HMW-CK).
2- Low molecular weight-CK (LMW-CK).
Pan-cytokeratin (AE1 & AE3)
* Cocktail of monoclonal antibodies to high and low
molecular weight cytokeratin.
* Recommended as primary reagent to identify
epithelial origin.

43. HMW-CK LMW-CK
* Belong to complex
epithelium & their tumors.
* e.g. ductal, transitional,
squamous carcinoma.
* Include CK 1 to 6 & 9 to 17
* Belong to simple
epithelium & their tumors.
* e.g. hepatocelluar,
pancreatic , colonic
carcinomas.
* Include CK 7, 8 , 18, 19,

44. Carcinomas classification
According to expression of low &/or high molecular weight cytokeratins,
carcinomas are classified into:-
Group I: HMW-CK+/LMW-CK+ carcinomas.
Group II: HMW-CK-/LMW-CK+ carcinomas.
Group III: HMW-CK+/LMW-CK- carcinomas.
HMW-CK-/LMW-CK- carcinomas: not exist.

45. HMW-CK+ LMW-CK+
Squamous cell
carcinoma
HCC
RCC
CRC
Neuroendocrine
Prostate
Breast
Pancreas
NSCLC
TCC
NSCLC: non small cell lung cancer
TCC: transitional cell carcinoma
HCC: hepatocellular carcinoma
RCC: renal cell carcinoma
CRC: colorectal carcinoma

46. CK7 & CK20 expression
According to expression of CK7 &/or CK20, tumors that express LMW-CK
can be further divided into 4 group:-
CK7+/CK20+ carcinomas: e.g. pancreatic
carcinoma.
CK7-/CK20- carcinomas: e.g. HCC.
CK7+/CK20- carcinomas: e.g. NSCLC.
CK7-/CK20+ carcinomas: e.g. CRC.

47. CK7+/CK20
+* Transitional cell carcinoma.
* Pancreatic carcinoma.
* Ovarian mucinous carcinoma.
CK7-/CK20-
* Hepatocelluar carcinoma.
* Renal cell carcinoma.
* Prostatic carcinoma.
* Squamous cell carcinoma.
* Neuroendocrine carcinomas.
CK7+/CK20-
* Breast carcinoma.
* NSCLC.
* Thymoma.
* Endometrial adenocarcinoma.
CK7-/CK20+
* Colorectal carcinoma.

48. CK20CK7Carcinoma type
++Transitional cell carcinoma
+/-+/-Pancreatic carcinoma
+/-+/-Ovarian mucinous carcinoma
CK20CK7Carcinoma type
----Hepatocellular carcinoma
----Renal cell carcinoma
----Prostatic adenocarcinoma
----Squamous cell carcinoma
--+/-Neuroendocrine carcinoma
CK20CK7Carcinoma type
--+Breast duct carcinoma
--+NSCLC
--+Thymoma
--+Salivary adenocarcinoma
--+Ovarian serous carcinoma
--+Ovarian endometrioid carcinoma
--+Endometrial carcinoma
--+Endocervical carcinoma
CK20CK7Carcinoma type
+--Colorectal carcinoma
+--Merkel cell carcinoma

51. CK7
Multifocal lesions in liver ??
+ve
-ve
CK20
CK20
+ve
+ve
-ve
-ve
Pancreatic carcinoma
Breast or NSCLC
Colorectal carcinoma
Hepatocellular carcinoma

52. CK7
Multiple nodules in lung??
+ve
-ve
CK20
CK20
+ve
+ve
-ve
-ve
TCC
Ovarian mucinous
Pancreatic
NSCLC or Breast
Endometrial
Colorectal carcinoma
Squamous cell carcinoma
HCC, RCC, Prostate
Neuroendocrine

53. Cytokeratin co-expression
A group of sarcomas characterize by expression of
cytokeratin in addition to vimentin expression:-
1- Synovial sarcoma.
2- Epithelioid sarcoma.
3- Mesothelioma.
4- Chordoma.

54. Carcinoembryonic Antigen (CEA)
= CD66
Demonstrated in
Variable degrees in adenocarcinomas.
CEA family
1- True CEA.
2-NCA (nonspecific cross-reacting antigen).
3-BGP (biliary glycoprotein).

55. Role
1- Identify primary site as most common carcinomas
that express CEA are adenocarcinoma of lung,
colorectal carcinoma, HCC (canalicular pattern).
2- CEA expression usually directly proportional to
degree of cellular differentiation >> well
differentiated express more CEA than poorly.

56. CEA-positive
carcinomas1- NSCLC.
2- Colorectal carcinoma.
3- Hepatocellular carcinoma.
CEA-negative carcinomas
1- Mesothelioma.
2- Renal cell carcinoma.
3- Ovarian carcinoma.
4- Endometrial carcinoma.
CEA-positive or negative
carcinomas
1- SCLC
2- Breast carcinoma.
3- TCC.
4- Cervical carcinoma.

57. Hormone Receptors (ER & PR)
Positive ER &
PR
1- Breast carcinoma.
2- Ovarian carcinoma.
3- Endometrial carcinoma.
4- Cervical carcinoma.
5- Sweat gland.
6- Thyroid carcinoma
7- Carcinoids.
Negative ER &
PR
1- NSCLC.
2- Colorectal carcinoma.
3- HCC.
Positive reaction could
be not considered
specific for metastatic
breast carcinoma

58. Specific carcinoma markers
Prostatic Specific Antigen (PSA)
Prostatic Acid Phosphatase (PAP)
Gross Cystic Disease Fluid
Protein-15 (GCDFP-15)
Thyroglobulin
Thyroid Transcription Factor-1
(TTF-1)
Uroplakin III
HepPar-1, GPC-3
CDX2
Prostate carcinoma
Prostate carcinoma
Breast carcinoma
Thyroid carcinoma
Thyroid carcinoma
Urothelial carcinoma
Hepatocellular carcinoma
Colon carcinoma

59. Prostatic Acid Phosphatase (PAP)
* Non specific for prostatic carcinoma.
* Expressed also in hindgut carcinoids.

60. Prostate Specific Antigen (PSA)
* Specific for prostatic carcinoma.
* In combination of PAP, identify 95% of all
metastatic prostatic carcinoma.

61. Gross cystic Disease Fluid Protein-15
(GCDFP-15)
* Highly Specific for breast carcinoma.
* 50% sensitivity.

62. Thyroglobulin (TG)
* Specific and sensitive markers for both primary
and metastatic thyroid carcinomas.

63. Thyroid Transcription Factor-1 (TTF-1)
* Immunoreactive to thyroid carcinomas.
* 70% of NSCLC.

64. Uroplakins
* Transmembrane proteins
* Expressed by urothelial umbrella cells.
* Moderate sensitivity & highly specific marker of
transitional cell carcinoma.

67. Sarcoma Markers
1- General markers:-
* Vimentin
2- Specific sarcoma markers.
* Vascular markers (CD31, CD34, Factor VIII).
* Muscle markers (Actin, Myosin).
* Bone markers (Osteonectin, Osteocalcin).
* CD68.

68. Vimentin
Definition
Intermediate filament of mesenchymal tumors (soft tissue
and bone sarcomas).
Vimentin co-expression
Carcinomas that express vimentin in addition to cytokeratin
expression, mostly of mesenchymal origin.

69. Carcinomas of mesenchymal origin
1- Adrenal cortical carcinoma.
2- Renal cell carcinoma.
3- Epithelioid mesothelioma.
4- Ovarian & endometrial carcinoma.
5- Müllerian & mesonephric carcinoma.
6- Bladder trigone carcinoma.
7- Central prostate carcinoma.

70. Common Vimentin
co-expression
1- Renal cell carcinoma.
2- Endometrial carcinoma.
3- Salivary gland carcinoma.
4- Sweat gland carcinoma.
5- Mesothelioma.
6- Thyroid carcinoma
7- Spindle cell carcinoma.
Rare Vimentin
co-expression
1- Breast Carcinoma.
2- Ovarian carcinoma
3- NSCLC.
4- SCLC.
5- Prostate carcinoma
6- Colorectal carcinoma.
7- Hepatocellular carcinoma.

71. Other non-sarcomatous tumors that express vimentin
1- Malignant melanoma.
2- PNET.
3- Neuroendocrine tumors.
4- Gonadal Germinomas.
5- Langerhans cell tumors

72. Vascular Markers
1- CD31.
2- Factor VIII related antigen.
3- CD34.
4- Ulex lectin.

73. CD31
Definition
Endothelial cell adhesion molecule.
Expression
* Angiosarcoma (78%).
* Kaposi’s sarcoma (100%).
Advantage
Highly sensitive marker.

74. Factor VIII related antigen
Expression
Benign & malignant endothelial cells.
Disadvantage
Low sensitivity

75. CD34
Expression
* Angiosarcoma (70%).
* Kaposi’s sarcoma (90%).
* Hemangioendothelioma (100%).
Disadvantage Non specific as its also present in
* GIST.
* Dermatofibrosarcoma protuberance.

76. Muscle Markers
1- Desmin.
2- Myoglobin.
3- Myogenin.
4- Actin.
5- Myosin.

77. Desmin
Definition
Sensitive marker of myosarcomas (smooth & skeletal muscle
origin).
Expression
* Rhabdomyosarcoma (90%).
* Leiomyosarcomas (variable): uterine > extra-uterine.

78. Muscle-specific Actin (MSA)
Specific marker for skeletal & smooth muscle phenotype
(Rhabdomyosarcoma, leiomyosarcoma).

79. Myoglobin
Specific marker for skeletal muscle phenotype
(Rhabdomyosarcoma).

80. Myogenin
Specific marker for skeletal muscle phenotype
(Rhabdomyosarcoma).

81. MyoD1
Specific marker for skeletal muscle phenotype
(Rhabdomyosarcoma).

82. Smooth Muscle Actin (SMA)
Specific marker for smooth muscle phenotype
(leiomyosarcoma).

83. Smooth Muscle Myosin (SMM)
Specific marker for smooth muscle phenotype
(leiomyosarcoma).

84. CD68
Lysosomal glycoprotein.
Definition
Expression
Histocytes.
Positive in
50-90 % of malignant fibrous histiocytoma.
Drawbacks
Positive in reactive histiocytes also.

86. Neuroectodermal tumors Markers
1- General markers:-
* Neuron-Specific Enolase (NSE).
* Chromogranin.
* Synaptophysin.
* S100-protein.
* Leu-7.
* Neurofilament protein (NF).
2- Specific NET markers.

87. Neuron-Specific Enolase (NSE)
* Very sensitive.
* Non-specific marker as non neuroectodermal
tumor is positive for NSE e.g. adrenal cortical
tumors.

88. Chromogranin
* Specific marker as it represents secretory granules
of these tumors.
* 3 types:-
1- Chromogranin A.
2- Chromogranin B.
3- Scretogranin II (sg II).

89. Synaptophysin
* A component of membrane of presynaptic vesicle.

90. S-100 protein
* Very sensitive.
* Non-specific marker as many non
neuroectodermal tumor is positive for S-100.
* Malignant melanoma (95%) express S-100 protein.
* Paragangliomas: S-100 positivity is limited to sustentacular
stromal cells.

91. Non-NET positive for S-100 protein
1- Chondrosarcoma.
2- Liposarcoma.
3- Leiomyosarcoma.
4- Synovial sarcoma.

92. Leu-7
Monoclonal antibodies that was produced against
T-cell leukemia & founded to be reactive against
NET.

93. Specific neuroectodermal tumors markers
Astrocytoma
Melanoma
Ependymoma
Neurofibroma
Pineocytoma
Meningioma
GFAP: Glial Fibrillary Acidic Protein
EMA: Epithelial Membrane Antigen
HMB-45
GFAP
GFAP
Neurofilament (NF)
Neurofilament (NF)
EMA, CK, Vimentin
Peripheral PNET CD99

94. Specific neuroendocrine tumors markers
Pheochromocytoma
Neuroblastoma
GI carcinoids
SCLC
Pancreatic islet
Thyroid C-cell
ACTH: Adrenocorticotropin
PP: pancreatic polypeptide
VIP: vasoactive intestinal polypeptides
Catecholamines
Encephalins, Somatostatin
Serotonin
Ectopic ACTH
Insulin, Glucagon, Gastrin, PP,
VIP
Calcitonin

95. CD99 (MIC-2)
Surface glycoprotein (membranous staining pattern.
Definition
Expression
Peripheral primitive neuroectodermal tumors (Ewing/PNET).
Limitations
1- Negative in central PNET e.g. medulloblastoma.
2- Positive in other tumors.

96. Non-PNET positive for CD99 (cytoplasmic)
1- Lymphoma.
2- Rhabdomyosarcoma.
3- Malignant Fibrous Histiocytoma (MFH).
4- Mesothelioma.
5- Synovial sarcoma.

98. Germ cell tumors Markers
1- General markers:-
* Placental Alkaline Phosphatase (PLAP)
2- Specific germ cell tumors markers.

99. Placental Alkaline Phosphatase
(PLAP)
1- Germinoma: highest (98%), accentuated membranous
pattern.
2- Embryonal carcinoma: high (86-97%).
3- Choriocarcinoma: low (about 50%).
4- Yolk sac tumor: low (about 50%).
Not specific: 13% of carcinomas are positive for PLAP

100. Specific germ cell tumors markers
Embryonal carcinoma
Seminoma
Yolk sac tumors
Choriocarcinoma
Sex cord tumors
CK8+, CK18+
CK+ , EMA- , AFP+ (low rate)
AFP+ (high rate, patchy)
B-HCG (synctio & intermdeate
trophoblasts), CK8+, CK18+,
EMA+
Inhibin

102. Hemolymphoid tumors markers
1- General markers:-
* Leucocyte Common Antigen (LCA) = CD45.
* CD19 & CD20 >>>> B-phenotype.
* CD3 & CD45RO >>>> T-phenotype
2- Specific Hemolymphoid tumors markers.

103. Histogenesis of lymphoma/leukemia. A mother stem cell gives rise to T,
Natural killer NK and B-cells. Hodgkin lymphoma and myeloma are of
B-cell origin.

104. Specific Hemolymphoid tumors
markers
(I) Hodgkin's Lymphoma markers
Lymphocyte predominant CD45+, CD20+
Classic types CD15+, CD30+
B-
lymphocytes
phenotype
Monocyte
phenotype

105. (II) Non-Hodgkin's Lymphoma markers
1- B-cell NHL (CD20+):-
* Small B-cell NHL.
* Large B-cell NHL.
2- T-cell NHL (CD3+):-
* Large T-cell NHL.

106. CD5
Small B-cell NHL (CD20+)
+ve
-ve
CD2
3
CD10
+ve
+ve
-ve
-ve
Small lymphocytic
lymphoma (SLL)
Mantle Cell Lymphoma
(MCL)
Follicular lymphoma
(FL)
Mantle Zone Lymphoma
(MZL)

107. B-cell lymphoma markers
B-CLL
B-Lymphoblastic
Lymphoplasmacytoid
Plasmacytoma
Hairy cell leukemia
CD20+, TdT+
CD20+, CD5+, CD23+
CD20+, CD5-, CD10-, CIg+
CD20-, CD38+, PCA-1+, CIg+
CD20+, CD5-, CD10-, CD23-,
CD11c+, CD25+
Marginal zone CD20+,CD5-, CD10-, CD23-
Follicular CD20+, CD5-, CD10+, bcl-2+
Mantle cell CD20+, CD5+, CD23-, Cyclin D-
1+
Burkitt’s CD20+, CD5-, CD10+

108. CD30
Large T-cell NHL (CD3+)
+ve
-ve
Anaplastic large T-cell
lymphoma
Peripheral T-cell
lymphoma

109. T/NK-cell lymphoma markers
T-CLL
T-Lymphoblastic
Mycosis fungoides
Peripheral T-cell
Nasal type T/NK
CD3+, CD4+, CD8+, TdT+
CD3+, CD4+
CD3+, CD4+, CD5+
CD3+, CD4+, CD5+, CD8+
CD3+, CD2+/-, CD56+
Anaplastic large cell CD3+,CD30+, EMA+/-
Adult T-cell lymphoma/leukemia CD3+, CD4+

110. (III) Myeloma markers
* CD19-
Positive
* CD38
* CD43
* CD56
* CD138
* CD79a
Negative
Occasionally +ve
* CD117
* Cyclin D-1
Occasionally -ve
* CD20
* CD45

111. (IV) Histiocytic tumors markers
* CD21
* CD35
Histiocytic cells
* CD68
* CD11c
Dendritic cells
Langerhans cell histiocytosis
Non Langerhans cell histiocytosis
Follicular dendritic sarcoma
S-100, CD1a, CD207
(Langerin)
CD68, CD163, cathepsin B
CD21, CD23

112. Immunocytology
(Flow-cytometry)

113. Definition
Identification of membrane and intracytoplasmic CD
antigens on and in different cell types using specific
antibodies.

114. Indications
• Acute leukemias: essential for primary diagnosis; follow-up only with
“informative phenotype”.
• Lymphomas: non-Hodgkin’s lymphomas, hairy cell leukemia,
plasmacytoma.
• Myeloproliferative syndrome (MPS)/myelodysplastic syndrome (MDS):
characterization of blasts in the development of acute leukemia
• Organ infiltration by epithelial tumor cells: detection of cytokeratin-positive
cells. he prognostic value of “minimal tumor infiltration” (TNM stage M(i))
of the bone marrow is under debate
• Cellular immune defect constellations.
• Quantification of hematopoietic progenitor cells (CD34+).

115. Suitable samples
• Peripheral blood.
• Lymph node aspirate or biopsy.
• Bone marrow aspirate.
• Body fluids.
• Liquor.
• Other aspirates, e.g., from skin.
Advantage
• Can be carried out on a limited number of cells.
• Larger antibody panel can be used than IHC.

131. Cytogenetics

132. Definition
Methods of detecting clonal chromosomal aberrations
in malignant cells important for primary diagnosis,
assessment of progression, therapy, and prognosis of
hematological diseases
Include
1- Classic Cytogenetics (Karyotyping).
2- FISH.

133. Clonal Chromosomal aberrations (abnormalities)
1- Primary disease-specific abnormalities e.g., t(9;22),
(“Philadelphia chromosome,” sole chromosomal abnormality
in the chronic phase of CML), pathogenetically of causal
significance.
2- Secondary chromosomal abnormalities in connection with
genomic instability and clonal evolution (e.g., multiple,
unspecific structural aberrations), pathogenetically of no
causal significance.
Clonal criteria
* Evidence of an identical structural chromosome abnormality or additional
chromosome in =>2 cells
* Evidence of identical numeric chromosome abnormality in > 3 cells

134. Objective
Detection of numerical and structural chromosome
aberrations in malignant cell clones.
Karyotyping

135. Indications
• Primary diagnosis: acute leukemia (AML, ALL), myelodysplastic syndrome
(MDS), chronic myeloid leukemia (CML) and other myeloproliferative
syndromes (MPS), multiple myeloma (MM), chronic lymphatic leukemia
(CLL).
• Post-therapeutic follow-up: only if a cytogenetic marker has been identified
and other diagnostic methods (morphology, Immunocytology, molecular
diagnosis with PCR) do not yield clear results.
• Evaluation of prognosis: for AML, MDS, ALL, MM, CLL, CML (see above).
• Progression or transformation of hematological diseases (e.g., MDS,

136. Limitations
• Tests dependent on availability of sufficient cell material (ideally first
marrow aspirate) and conditions of sampling and dispatch (sterility) risk of
false-negative results due to insufficient material or < 10 analyzable
metaphases.
• Even if sufficient material, sensitivity is 1:20 to 1:30 due to limited number
of analyzable cells, therefore detection of minimal residual disease (MRD)
not possible when < 5% of cells show cytogenetic marker.
• Tests dependent on cell division a normal karyotype does not rule out
abnormal, non-dividing clones.
• Submicroscopic structural aberrations non-detectable.

137. Nomenclature

138. Examples

139. Objective
Detection (quantitative) of known numerical or
structural abnormalities, especially in follow-up
Examinations.
Fluorescence In Situ
Hybridization (FISH)

140. Indications
• Primary diagnosis: acute leukemia (AML, ALL), myelodysplastic syndrome
(MDS), chronic myeloid leukemia (CML) and other myeloproliferative
syndromes (MPS), multiple myeloma (MM), chronic lymphatic leukemia
(CLL).
• Post-therapeutic follow-up: only if a cytogenetic marker has been identified
and other diagnostic methods (morphology, Immunocytology, molecular
diagnosis with PCR) do not yield clear results.
• Evaluation of prognosis: for AML, MDS, ALL, MM, CLL, CML (see above).
• Progression or transformation of hematological diseases (e.g., MDS,
(especially in case of lack of significance of classic cytogenetic methods)

141. Advantages Over Classic
Cytogenetics
• Detection limit: 100–1,000 cells can be analyzed higher sensitivity
• Interphase-FISH analysis does not depend on cell division and cell culture
variations, hence allowing quantitative conclusions compared with
“classic” metaphase cytogenetics.
• Suitable for follow-up tests with established cytogenetic markers.
• Lower demands on quality regarding sampling and dispatch.
• Conclusions about aberrations in diseases with otherwise unsuccessful
karyotyping (e.g., MDS with marrow fibrosis, Hypocellular AML).

142. Limitations
• Specificity: only known or presumed numerical or structural
aberrations which complement the used DNA probe can be
detected (not a global test for the detection of all
chromosomal aberrations), hence supplementary to
chromosome analysis
• Quality of the used DNA probe

143. Molecular diagnosis

144. Definition
Detection and characterization of genetic and
epigenetic alterations associated with malignancies.
Specific nucleic acid modifications serve as molecular
markers for malignant cell clones.

145. Indications
• Confirmation of diagnosis via detection of tumor-associated
molecular markers.
• Identification of prognostically relevant subgroups / genotypes
within a tumor entity for therapy planning.
• Detection of minimal residual disease in the framework of
follow-up tests to allow early therapeutic intervention.

146. Examples

147. Method
• Isolation of DNA or RNA (depending on indication and marker).
• RNA-based assays: reverse transcription of RNA into cDNA.
• Amplification of DNA or cDNA via polymerase chain reaction (PCR) using
specific oligonucleotides (primer).
• Quantitative analysis via “real-time” PCR of the amplification products;
semiquantitative analysis via gel electrophoresis (agarose,
polyacrylamide)
• In specific cases: analysis of genomic DNA (Southern blot), RNA analysis
(Northern blot), ligase chain reaction, and other methods

148. Advantage
• Only small amounts of material required for analysis; no specific fixation
necessary.
• High sensitivity of PCR-based methods; particularly suitable for the
detection of minimal residual disease (1 cell/1000000 cell).
• Compared with cytogenetics, no need for proliferating cells.
Disadvantage
• Formalin-fixed samples less suitable due to degradation of nucleic acids.
• Analysis of only one molecular marker per assay.
• Rigorous quality controls and intricate isolation are necessary measures
due to the high sensitivity of PCR-based assays >>> contamination with
foreign material would yield false-positive results.

149. Conformation of diagnosis
• CML, myeloproliferative syndromes: BCR/ABL fusion gene.
• Malignant lymphoma: clonality of antigen receptor gene rearrangements.
• Follicular lymphoma: Ig/BCL2 rearrangement, t(14;18), t(2;18), t(18;22).
• Microgranular variant of acute promyelocytic leukemia (AML M3v):
PML/RAR-alpha.

150. AML (Primary Diagnosis): Identification of
Prognostically Relevant Subgroups
• AML1/ETO fusion gene: t(8;21)(q22;q22).
• CBF-beta/MYH11 fusion gene: inv(16) and t(16;16)(p13;q22).
• PML/RAR-alpha fusion gene: t(15;17)(q21;q22) with AML FAB M3.
• flt-3 mutations: internal tandem duplication, TK domain, mostly with normal
karyotype.
• NPM1 mutation: mostly with normal karyotype.

151. ALL (Primary Diagnosis): Identification of
Prognostically Relevant Subgroups
• BCR/ABL fusion gene: t(9;22)(q34;q11)
• Translocations of the MLL gene: e.g. AF-4/MLL: t(4;11)(q21;q23)
• E2A/PBX fusion gene: t(1;19)(q23;p13)

152. Soft tissue sarcoma (Primary Diagnosis): Identification
of Prognostically Relevant Subgroups
• Ewing’s sarcoma/PNET: EWS/FLI1 fusion gene: t(11;22)(q24;q12)
• Clear cell sarcoma: EWS/ATF fusion gene: t(12;22)(q13;12)
• Synovial sarcoma: SYT/SSX fusion gene: t(x;18)(p11;q11)
• Liposarcoma: TLS/CHOP10 fusion gene: t(12;16(q13;p11)
• Alveolar rhabdomyosarcoma: PAX3/FKHR fusion gene: t(2;13(q35;q14)

153. Minimal Residual Disease (MRD)
• CML: BCR/ABL fusion gene (especially in cytogenetic complete response:
imatinib or IFN-alpha treatment following allogeneic or autologous
hematopoietic transplantation, administration of donor lymphocytes).
• AML: depending on genotype at initial diagnosis (e.g., AML1/ETO, CBF-
beta/MYH11, PML/RAR-alpha).
• ALL: depending on genotype at initial diagnosis (especially with Ph1-ALL,
but after translocations of MLL gene, E2A/PBX), detection of clonotypical
antigen receptor gene rearrangement.
• NHL: depending on genotype at initial diagnosis (e.g., Ig/BCL2), detection
of the clonotypical antigen receptor gene rearrangement.

154. Gene Expression
Analysis (Microarrays)

155. Definition
Simultaneous surveying of the expression of
numerous defined genes of the human genome using
microarray technology (biochips). he human genome
consists of approximately 40,000 chromosomal genes.

156. Microarray
Hybridization of fluorescence-marked tumor RNA with defined
genetic probes on a glass chip.
• The number of probes varies from several hundred (low-
density chips) to several thousand (high-density chips),
depending on the chip. Probes consist of PCR-amplified
cDNA fragments (100–3,000 base pairs) or oligonucleotides
(25–80 base pairs).
• Hybridization of the sample RNA with the cDNA probe is
indicated by a fluorescence signal.
• Hybridization results (indicating gene expression patterns) are
obtained by automated scanning of the microarray chip.

157. Data Analysis
The large amount of data collected (with expression patterns of
up to several thousand genes) requires automated evaluation
procedures. Analysis of numeric gene expression is carried out
using complex mathematical algorithms, e.g. cluster analysis.
Cluster analysis results are presented as:
• Dendrograms (Cluster Trees) : tree-like presentation of gene
groups with similar expression patterns (cluster).
• Heat Maps: colored matrixes categorized into clusters that
indicate gene expression levels of differentially expressed
genes by different shades of color.

158. Indications
Elucidation of genetic contexts in tumor development and progression in
experimental model systems. Identification of tumor-specific targets forms the
basis for the development of targeted therapies
1- Identification of molecular mechanisms of tumor
development
2- Diagnosis and Prognosis of Malignancies
Global gene expression analysis allows for advanced classification and prognosis
evaluation of human neoplasias. In the future, these findings might have an
influence on therapeutic decisions. Clinical studies have proved the importance
of genetic profiles (“molecular signature”)
for:
• Acute leukemias: subtyping and risk classification.
• Diffuse large cell B-NHL: subtyping and risk classification.
• Breast cancer: risk classification.

159. Gene expression analysis permits predictions about the effectiveness and
resistance of pharmaceuticals, which could form the basis for future
individualized hematological and oncological therapy
• The analysis of 95 genes possibly allows for the prediction of chemosensitivity
or chemoresistance of imatinib therapy in Ph+ CML and ALL.
3- Pharmacogenomics

160. Advantage
• Large amount of information due to parallel analysis: mapping
of the entire transcriptome of a cell population.
Disadvantage
• High costs of high-density chips.
• Large amounts of data necessitating complex bioinformatic
analyses.
• For the majority of tumor types, target genes, expression
profiles and prognostic significance have not been
established.