general pathology

1. Guide: Dr. K.K.Kulkarni

2. contents
 Introduction
 Definition
 Historical background
 Properties of Ideal TM
 Classification
 Clinical applications
 Salient features of Individual TM
 Methods of detections
 TM & associated cancer
 Recent advances

3. Introduction
 A major challenge in the management of
patients with cancer is the lack of specific tools
for
• The early detection
• Accurate prediction of biological behavior
• Accurate assessment of prognosis.

4. Role of a Tumour marker
Tumor markers can play a crucial role in
detecting disease and assessing response to
therapy…

5. Definition
 Any cell product , viz, enzymes, serum proteins,
metabolites, receptors, oncoproteins, oncofoetal
proteins , can be used as a marker to diagnose the
presence of a malignancy, as long as it is related to
any event during tumour formation, proliferation,
dedifferentiation, etc.
 A substance present in or produced by a tumor
OR
A substance produced by the host in response to
the tumor’s presence

6. Historical background
 1846 – Bence jones first identified BJ protein in
urine of patients of MM
 1930-1990- Acid phosphatase served as marker
for prostate cancer
 1960 – radioimmunoassay
 1963 – AFP & 1965- CEA discovered as tumour
marker for HCC & colon cancer respectively.
 1975- monoclonal Abs tech was developed which
facilitates the discovery of new tumor markers
including CA125, CA15.3, CA19.9

7. Historical background cont….
 1980 – PSA Best cancer marker was discovered
 1970s & 1980s new concepts oncogenes and
tumor suppressor genes came into picture.
 2001- Microarrays, Mass Spectrometry, Neural
Networks, Multiparametric Analysis

8. Ideal tumour marker
 Highly sensitive
 Highly specific
 100% accurate in differentiating between healthy
individuals and tumor patients.
 Differentiate neoplastic and non-neoplastic disease
 show positive correlation with tumor volume and extent.
 Predict early recurrence and have prognostic value.
 Detectable at early stage of tumor
 Its levels should be preceding the neoplastic process.
 short half life to rapidly mirror treatment schedules
 Measured easily, reliably and cost-effectively using an
assay with high analytic sensitivity and specificity.

9. Classification
 Oncofetal antigens: AFP, CEA
 Tumor associated antigens : CA125, CA19-9, CA15-3,
CA72-4, CA50
 Hormones: β-hCG, Calcitonin
 Receptors : ER, PR, EGFR
 Enzymes and Isoenzymes : PSA, PAP, NSE, PALP,
 Serum and tissue proteins : -β2 microglobulin,
monoclonal immunoglobulin/para proteins, GFAP, protein
S-100, ferritin, fibrinogen
 Other biomolecules: polyamines
 Oncogenes: Ras, myc ,abl-bcr
 Tumour supressor genes: BRCA1 , P53, Rb

10. Clinical Applications

11. Role in Screening
 Tumor markers play a limited role for tumor
screening, just because….
 relatively low sensitivity
 lack of specificity
 Not elevated in early stage
 Inappropriate for the detection of small in situ cancer
 Examples Used as screening tool:
 AFP in liver cancer in China
 Urinary VMA and homovanillic acid for neuroblastoma in
Japanese children
 PSA along with DRE for prostate cancer in >50 yr aged men

12. Role in Diagnosis
 Most tumor marker levels alone are often insufficient
to diagnose cancer for the following reasons:
 TM levels can be elevated in people with benign conditions;
 TM levels are not elevated in every person with cancer,
especially in the early stages of the disease;
 Many TM are not specific to a particular type of cancer;
 The level of a TM can be elevated by more than one type of
cancer
 Tumor marker is not the key diagnostic tool, but can be a
complementary sign to clinical finding & medical imaging
 AFP & HCC

13. Diagnosis cont…
 Several approaches have been suggested
recently to improve the diagnostic yield of many
tumor markers:
 The use of multiple markers.
 By improving both the specificity and sensitivity of a
tumor markers, as in the case of serum PSA test,
involves the measurement of the velocity & the density,
complex PSA level , %fPSA

14. Role in staging/prognosis
 The pre-therapeutic level of certain tumor marker can
contributes a prognostic factor because of links with...
 Metabolic activity
 Tumor size
 Invasion
Eg:
-High PSA preoperatively associated with high gleason score, positive
surgical margins, + lymph node status
- ER +ve breast tumors have good prognosis
- C-erb-2-gene(HER-2/neu) is prognostic for ovarian & breast cancer
 Allow doctors to refine therapeutic strategy by
selecting groups with risk of failure response to
treatment

15. Monitoring & recurrence
 One of the most useful application
 The serum level of tumor markers reflects the success of surgery or
the efficacy of chemotherapy.
 If the marker level in the blood goes down, that is almost always a
sign that the treatment is effective
 If marker level after surgery remain elevated would indicate either
incomplete removal of the tumor, recurrence, or the presence of
metastases.
 The measurement of serum tumor markers during chemotherapy
also gives an indication of the effectiveness of the antitumor drug
used and a guide for the selection of the most effective drug for each
individual case.
 Eg: PSA for prostate cancer,
HCG for gestational trophoblastic tumours & germ cell tumours of
ovaries & testicles
CA 125 for epithelial ovarian cancer

16. Recommendations for ordering TM tests:
 Never rely on the results of a single test
 When ordering serial testing be certain to order
every test from the same laboratory using the same
kit
 Be certain that the tumour marker selected for
monitoring recurrence was elevated prior to the
surgery
 Consider half life of tumour marker when
interpreting the result
 Consider how the tumour marker is metabolized
or removed from the circulation

17. Recommendations for ordering TM tests:
 Consider ordering multiple markers to improve
sensitivity and specificity
 Order the non specific markers for cost saving and
high sensitivity
 Be aware of the possibility of hook effect and lastly,
 Be aware of possibility of ectopic markers

18. Biochemical markers
Oncofetal
antigen
Reference range Cancers
Elevated in
other benign
cond.
Application Sample
AFP < 10-20ng/ml HCC,
Germ cell cancer
of ovaries or
testes
Also elevated
during
pregnancy,
benign liver
disease
Diagnosis.
prognosis
,monitor
treatment,
and
determine
recurrence
Blood
CEA < 5
ng/ml
Colorectal, lung,
breast, gastric,
pancreatic, liver,
Elevated in
hepatitis,
COPD, colitis,
pancreatitis,
and in
cigarette
smokers
Monitor
treatment
and
determine
recurrence
Blood

19. Carbohydrate
antigen
Reference range Cancers
Elevated in other
benign cond.
Application Sample
CA-125 < 35U/ml Ovarian endometriosis,
Hepatitis,
cirrhosis
Early pregnancy
Help
diagnose,
Prognosis
monitor
treatment,
and
determine
recurrence
Blood
CA 15-3 < 30 U/ml Breast,
adenocarcino
ma of colon,
lung ovary,
pancreas
cirrhosis, acute &
chronic hepatitis,
Benign breast ds,
SLE
sarcoidosis
Monitor
treatment &
disease
progression
in metastatic
breast ca.
for follow up
to detect
recurrence
Blood

20. Carbohydra
te antigen
Reference range Cancers
Elevated in other
benign cond.
Application Sample
CA 27.29 < 0-38U/ml Breast,
adenocarcino
ma of colon,
lung, ovary,
pancreas
endometriosis,
Hepatitis,
cirrhosis
Early pregnancy
useful for
predicting
early
recurrence
in treated
breast pt.
Blood
CA 19-9 < 30 U/ml Pancreatic,
colorectal and
bile ducts
Pancreatitis and
inflammatory bowel
disease
Stage
disease,
monitor
treatment,
and
determine
recurrence
Blood

21. Carbohydra
te antigen
Reference range Cancers
Elevated in other benign
cond.
Application Sample
CA 72-4
(TAG-72)
< 4 U/ml Gastric,
lung, ovary,
colorectal
No evidence that it is
better than CA-125
but may be useful
when combined with it
Monitoring
treatment,
Blood

22. Hormones Reference range Cancers
Elevated in other
benign cond.
Application Sample
β HCG < 0-38U/ml Trophoblastic
&
Testicular
Elevated in
pregnancy,
testicular
failure
diagnose,
Monitor
treatment,
and
Determine
recurrence
Blood
Urine
In CSf
To
detect
Mets in
brain
Calcitonin < 10 ng/ml Medullary
thyroid ca
Bronchogenic ca
Pernicious anemia,
Thyroiditis
Help
diagnose,
monitor
treatment,
and
determine
recurrence
Blood

23. Enzymes Reference range Cancers Other remarks Application Sample
PSA < 50 yr- <2.5ng/ml
> 50 yr -< 4 ng/ml
fPSA/PSA > 0.19
Prostate BPH
Prostatitis
With Age
Screening
and help
diagnosis,
monitor
treatment,
and
determine
recurrence
Blood
NSE < 15ng/ml Neuroblastoma
small cell lung
cancer,
carcinoid,pheoc
hromocytoma &
other NE
tumour
Hemolytic
anemia,
Hepatic failure,
ESRD
Neurodegenrative
ds
Stroke
Brain injury
Monitor
treatment to
determine
prognosis
Blood
PAP another test for prostate cancer which was used before PSA test was developed.
It is rarely used now because PSA is much more sensitive

24. Protein Reference range Cancers Other remarks Application Sample
Β2m* 1000-2400 ng/ml Multiple
myeloma
CLL
lymphoma
Elevated in
CRF & dialysis
pt without
cancer
Determine
prognosis,
monitor
treatment
efficasy
Blood
TG < 25 ng/ml Thyroid Elevated in
grave’s ds,
Toxic nodular
gotre,
thyroiditis
Monitor
treatment ,
for follow up
to detect
recurrence
Blood
* CSF level useful to detect metastasis in CNS

25. Protein Reference range Cancers Other remarks Application Sample
Monoc
lonal
IG
Multiple myeloma
and Waldenstrom’s
macroglobulinemia
Overproduction
of an
immunoglobulin or
antibody, usually
detected by
protein
electrophoresis
Help
diagnose,
monitor
treatment,
and
determine
recurrence
Blood,
urine

26. Methods of detection
 Immuoassay by using monoclonal Abs
 RIA
 ELISA
 IHC: ER,PR, Her-2 neu
 FISH: Her-2 neu
 RT-PCR
 HPLC

27. Common Cancers
and
Associated popular TM

28. Bladder cancer
 At present, no urinary tumour markers are
recommended for bladder cancer screening.
 Bladder tumour antigen (BTA) and NMP22 can be
used along with cystoscopy for diagnosis and follow-up
although cystoscopy and urine cytology(gold
standard) are still considered the current standard.
Sensitivity
%
Specificity% Method of
detection
BTA 55-70% 70-75% EIA
NMP-22 80-85% 75-80% ELISA
Cytology 30-40% > 90%
Sensitivity is higher but specificity is lower than cytology

29. 29
NegativePositive
Control
Test
identify urinary
NMP22 levels
> 10 U/ml

30. Breast cancer
 At present, no tumour marker has been found to be
useful for screening or for diagnosis of early stage
breast cancer.
 At the time of diagnosis, breast cancer tissue should
be tested for ER & PR, as well as HER2 antigen. These
markers provide information on how aggressive the
cancer can be and how likely it will respond to certain
treatments.
 Methods – IHC, FISH, CISH, RT-PCR

31. Her 2 receptor overexpression detection

32. Breast cancer cont….
 CA15-3 and CA27.29:
 used to monitor therapy in Pt with advanced breast cancer .
 No role in screening & detection of early breast cancer.
 CA 27.29 has better sensitivity and specificity than CA 15-3
 One study - 27.29 was highly specific and sensitive in detecting
preclinical metastasis, and this may lead to prompt imaging of
probable sites of metastasis, possibly decreasing morbidity because of
earlier treatment.
 FDA approved CA 27.29 for detection of recurrent breast cancer in Pts
with stage II & or stage III disease.
 Method: ELISA using monoclonal Abs
RIA

33. Colorectal cancer
 At present, neither CEA nor CA19-9 is useful as a screening
test for colorectal cancer.
 An elevated CEA level before surgery may indicate worse
prognosis. If all the cancer has been removed, CEA should
return to normal levels in about 4 to 6 weeks after
treatment.
 CEA measurement every 3 to 6 months should be
considered to help early diagnosis of recurrence.
 CEA is also used to monitor patients being treated for
advanced or recurrent disease.
 If CEA is not elevated in patients with advanced or
recurrent disease, CA19-9 may be used to follow the
disease.

34. Liver cancer
 Periodic screening by serum AFP measurement and
ultrasound for chronic hepatitis carriers are useful to
detect liver cancer at early stage.
 AFP can also be used to follow up patients after
treatment

35. Lung cancer
 At present, no TM have been proven to be useful as
screening tests .
 Tumour markers that can be raised in lung cancer
include CEA in non-small cell lung cancer and NSE in
small cell lung cancer.
 Because lung cancer is usually visible on CXR or other
imaging studies, TM play a less important role in
follow-up.

36. Prostate cancer
 PSA is commonly used to detect prostate cancer at early stage.
 Levels above 4ng/ml suggest cancer whereas levels above 10
ng/ml strongly suggest cancer.
 PSA is very useful in follow-up. After curative surgery, PSA level
should be zero or very close to zero. Those treated with
radiotherapy should also have a significant drop in PSA after
treatment.
 A subsequent rise in PSA after treatment could indicate relapse.
 PSA can also be used to assess response to treatment for advanced
disease.
 In rare cases, prostate cancers that do not have raised PSA levels
and do not respond well to hormonal therapy may turn out to
have neuroendocrine features. Patients with these cancers may
have higher levels of Chromogranin A.

37. Ovarian cancer
 CA125 is very effective to assess response of epithelial
ovarian cancer to treatment or to detect recurrence.
 CA125 can be used to screen for ovarian cancer in women
with strong family history of ovarian cancers. Such
women usually receive regular ultrasounds together with
CA125 measurements.
 Patients with ovarian germ cell tumours often have raised
levels of HCG and / or AFP, which are useful in diagnosis
and follow-up.

38. Melanoma skin
 At present, no tumour marker is of value in early
detection of melanoma.
 Tumour markers TA-90 and S-100 can be used to test
tissue samples to help diagnose melanoma.
 Serum level of S-100 is elevated when disease is
widespread. Thus it can be used to look for
progression of melanoma.
 Blood levels of TA-90 have been used to assess the
chance of metastasis of melanoma.

39.  Tumour markers commonly elevated in patients with
testicular cancer are HCG and AFP.
 Seminoma: About 10% of men with seminoma will
have raised HCG. None will have elevated AFP.
 Non-seminoma: More than half of men with early
stage disease have raised HCG or AFP or both. The
markers will be elevated in most men with advanced
disease.
 HCG is almost always raised and AFP is never elevated
in choriocarcinoma.
 In contrast, AFP but not HCG is raised in yolk sac
tumour or endodermal sinus tumour.
Testicular cancer

40. Disease Marker Screening
Detection
and
diagnosis
Staging and
prognosis
Follow
up
Sarcoma:
Synovial
sarcoma
t(X;18) X X
Ewing's
sarcoma
t(11;22) X
Alveolar
rhabdomyos
arcoma
t(2;13) X
Granolytic
sarcoma
t((9;11) X

41. Disease Marker Screening
Detection and
diagnosis
Staging
and
prognosis Follow up
Myxoid
liposarcoma
t(12;16) X
Round cell
liposarcoma
t(12;16) X
Congenital
fibrosarcoma
t(2;15) X
Clear cell
sarcoma
t(12;22) X

42. Disease Marker Screening
Detection and
diagnosis
Staging
and
prognosis Follow up
Dermatofibr
osarcoma
protuberans
t(17;22) X
Melanoma Tyrosinase X
Adrenal
carcinoma
Steroids X
Catecholami
nes
X

43. Disease Marker Screening
Detection and
diagnosis
Staging
and
prognosis Follow up
Lymphoma t(8;14) X X X
t(11;14) X X X
t(2;5) X X X
t(3;14) X X X
sCD25 X
sCD44 X
Leukaemia Numerous
cytogenetic
alterations
X X

44. Recent advances

46. Mechanisms for Induction of Carcinogenesis by
Mitogenic Pathway Elements
 Growth factors: a. Overproduction by cell into surroundings
b. Interaction of growth factors with high affinity receptors
 Growth factor a. Overexpression leading to high concentration of dimers
receptor b. Loss of extracellular domain resulting in permanent
dimerization of GFR and continuous signaling
c. Amino acid substitutions in transmembrane domain
leading to permanent dimerization
 Cytosolic proteins a. Overexpression of normal G-proteins and protein kinases
b. Amino acid substitutions that permanently change
conformation to activated form
c. Mutations that remove regulatory domains of kinases
 Nuclear oncoproteins a. Overexpression of transcription and replication proteins
b. Mutations in antioncogene proteins that inactivate them
c. Mutations that remove regulatory domains

47. Growth Factors
 Since various growth factors are believed to play a role in
influencing cellular proliferation during tumorigenesis
and since growth factors are actively secreted into the
extracellular environment, they are potentially attractive
targets for detection in blood during cancer
development.
 TGF α & β
 PDGF
 bFGF
 EGF & HGF

48. TGF α & β
 serum assays for TGF-β:
 useful for diagnosis and follow up in HCC
 Useful for diagnosis of invasive bladder cancer bcz of
high sensitivity and specificity
 TGF α:
 useful in screening the patients for the presence of
malignant tumours unlike TGF β not tumour specific
 Effective predictor of the occurrence of malignancies at
an early stage.
Eg. Elevated in people with asbestosis showing
progression to lung ca

49. PDGF
 Significantly elevated in over 15% of patients with
carcinomas, sarcomas, and lymphomas but not at all in
normal individuals.
 Excellent correlation between the serum level and the
stage of the breast cancer. Higher levels predict shorter
survival.
bFGF
 Markedly elevated in > 50% pateints of RCC
 Also elevated in the sera of over 50% of patients with CNS
tumors, 90% of patients with lung cancers and >60% of
patients with lymphomas
 Recently, elevated serum levels of bFGF have been found to
be a good prognostic factor in patients with non-small-cell
carcinomas of the lung.

50. Growth factor receptor
Transmembrane growth factor receptors : EGFr &
Her2/neu
 Encoded by the erbB family of oncogenes.
 Attractive targets for detection in blood during
cancer development. Extracellular domains of
these receptors enter circulation and can be
detected using immunoassays.

51. EGFr
• Elevations (> 636 fmol/mL) of circulating levels of ECD of
EGFr ,found in patients either have an asbestos-associated
malignancy or subsequently develop such malignancy.
• EGFr appears to be an excellent marker for asbestos-induced
tumors

52. Her 2/neu
 Elevated in the sera of patients with a number of different
epithelial cell cancers, including breast, lung, colorectal,
and ovarian cancers
 About 25% of patients with breast cancer over express
HER2, which is associated with aggressive disease, poor
clinical outcomes and shortened overall survival
 Samples of tumour tissue (not blood sample) are used to
test for HER2 status
 Clinical relevance:
 Correlates with poor prognosis
 Correlates with lower response to endocrine therapy
 Her2 positive tumors often have lost expression of ER/PR
 Predicts response to anthracyclines through linkage to TOPO2 gene, with
which it is often co-amplified
 Predicts response to targeted therapy (Trastuzumab/Herceptin)

53. P185 ECD of Her2/neu receptor
 Overexpression is accompanied by cleavage of the ECD of
the receptor with its accumulation in the extracellular
environment. Thus, detection of increased amounts of it in
blood is a potential biomarker of cancer development.
 In breast cancer : serum level correlate with tumor load
and stage. More sensitive ELISA assay for it, helpful in
detecting carcinoma in situ .
 In pulmonary cancer : Markedly elevated in Pt with
pnemoconioses prior to onset of frank malignancy
thus it is a highly sensitive marker for pulmonary cancer

54. P185 ECD of Her2/neu cont….
 Elevated in almost 100% of East Asian poeple who have
known risk factors for developing HCC. Thus good
sensitive marker like TGFβ.
 Direct correlation between serum levels of p185 ECD and
tumor size for premalignant adenomas of the colon .
Since colonic neoplasia usually progresses through well-
defined steps from adenoma to carcinoma with the
malignant potential of adenomas increasing with size,
serum erbB-2 ECD levels may be useful in monitoring
these progression.

55. G protein – cytosolic protein
gene encoded G protein
Normally transmit growth signals from cell membrane to cytosolic kinase
Overexpression AA substitution leading to mutated form
Uncontrolled cell division & carcinogenesis
Process is well documented for ras encoded p21 protein
Mutant p21 protein abnormal activation of alternate unregulated
signal transduction pathways
Gain access to extracellular environment by unexplained mechanisms
detected by immunoblotting with monoclonal antibodies.

56.  Elevated serum p21 is a biomarker of early malignant
disease in patients with a known predisposition.
 Known to be increased in patients with
pneumoconiosis with later progression to cancer.
 Mutated p21 found in serum of patients of
angiosarcoma with a known exposure to vinyl chloride.
there is direct correlation between duration of
exposure & occurrence of mutant p21.
 K-ras, mutation detected by molecular technology in
stool and lavage fluid, pancreatic and duodenal juices,
and sputum and lavage fluids for colorectal, pancreas
and lung cancers, respectively.

57. Nuclear oncoproteins
 Important nuclear oncoproteins :
 p53 tumour supressor gene protein
 p62/64 protein encoded by c-myc oncogene
 nuclear matrix proteins
 p53 – Antioncogene repress mitotic process & activate
proapoptotic protein which promote apoptosis in
transformed cells.
mutation in p53
Loss of normal growth inhibitory function
P53 proteins with increased half lives accumulate in cells
leak into extracellular environment

58.  Increased mutant p53 found in HCC & cirrhosis which
is risk factor of HCC. Thus elevated mutant p53 level in
cirrhosis patient is an early indicator of carcinogenesis.
 In breast cancer, p53 mutation is associated with more
aggressive disease and worse overall survival.
 Mutant p53 also found in shed cells in the urine of
bladder cancer patients so these noninvasive test can
be used for screening with other markers.
 Elevated levels of p53 antibodies are found in patients
with ovarian and colon malignancies, lung ca, breast
ca including intraductal ca.
 Anti-p53 antibodies found in patients with oral
premalignant lesions - marker for early detection of
oral ca.

59. C-myc encoded protein : P62
• overexpressed in burkitt’s lymphoma.
• C-myc protein elevated in the serum of patients of
breast ca & colon ca & marked diminution with
treatment. so useful in following the course of these
tumour.
• P62 overexpression seen in breast ca using IHC,
staining intensity greater with increased stage of
tumour.
Anti c-myc protein antibodies:
• Found in sera of pateints with colorectal cancer,
myeloid leukemia & burkitt’s lymphoma.

60. Nuclear matrix protein:
• NMP22 protein 10-20 times higher in malignant
transitional epithelial cells than normal cells. this the basis
for the detection of this protein into urine of bladder ca
patients by ELISA.
• Sensitivity in detection of malignant invasive cancer is 90%
& for ca in situ 75% specificity is 90%.
• Even higher sensitivity than cytology in Grade I, II,& III
TCC
• Clinical trial data showed that the NMP22 test, when
performed 6-40 days post-surgery, correctly predicted the
presence of recurrent disease.
• US FDA approved this method for screening & follow up of
bladder ca patient who have been treated for this disease.

61. summary
 Tumour markers may be used to help diagnose cancer,
predict and monitor response to treatment and
determine whether cancer has recurred after
treatment.
 In general, tumour markers alone cannot be used to
diagnose cancer, they must be combined with other
tests. Studies are being done to determine if tumour
markers can be used in early detection and diagnosis
of cancer.