Tumor Markers

Alan H.B. Wu, Ph.D.

Professor, Laboratory Medicine, UCSF
Section Chief, Clinical Chemistry, Toxicology,
Pharmacogenomics Laboratory, SFGH
 Learning objectives

• Know the ideal characteristics of a tumor marker

• Understand the role of tumor markers for diagnosis and
management of patients with cancer.
• Know the emerging technologies for tumor markers
• Understand the role of tumor markers for therapeutic
selection
 How do we diagnose cancer today?
Physical Examination
Blood tests
CT scans
Biopsy
Human Prostate Cancer

Normal Blood Smear Chronic Myeloid Leukemia

Death rates for cancer vs. heart disease
 New cancer cases per year
Cancer Site or Type New Cases
Prostate 218,000
Lung 222,500
Breast 207,500
Colorectal 149,000
Urinary system 131,500
Skin 68,770
Pancreas 43,100
Ovarian 22,000
Myeloma 20,200
Thyroid 44,700
Germ Cell 9,000
 Types of Tumor Markers

• Hormones
(hCG; calcitonin; gastrin; prolactin;)
• Enzymes
(acid phosphatase; alkaline phosphatase; PSA)
• Cancer antigen proteins & glycoproteins
(CA125; CA 15.3; CA19.9)
• Metabolites (norepinephrine, epinephrine)
• Normal proteins (thyroglobulin)
• Oncofetal antigens
(CEA, AFP)
• Receptors
(ER, PR, EGFR)
• Genetic changes
(mutations/translocations, etc.)
 Characteristics of an ideal tumor marker

• Specificity for a single type of cancer

• High sensitivity and specificity for cancerous growth
• Correlation of marker level with tumor size
• Homogeneous (i.e., minimal post-translational
modifications)
• Short half-life in circulation
 Roles for tumor markers
• Determine risk (PSA)
• Screen for early cancer (calcitonin, occult blood)
• Diagnose a type of cancer (hCG, catecholamines)
• Estimate prognosis (CA125)
• Predict response to therapy (CA15-3, CA125, PSA, hCG)
• Monitor for disease recurrence or progression (most widely
used function)
• Therapeutic selection (her2/neu, kras)
 Tumor markers in routine use
Marker Cancer
CA15-3, BR 27.29 Breast
CEA, CA 19-9 Colorectal
CA 72.4, CA 19-9, CEA Gastric
NSE, CYFA 21.1 Lung
PSA, PAP Prostate
CA 125 Ovarian
Calcitonin, thyroglobulin Thyroid
hCG Trophoblastic
CA 19-9, CEA Pancreatic
AFP, CA 19-1 Hepatocellular
BAP, Osteocalcin, NTx Bone
Catecholamines, metabolites Pheochromocytoma
Fecal occult blood Colon cancer
 Case report

• 38-y M complains of severe headaches, episodic, and

uncontrolled by analgesics.
• No hx of migranes. In clinic, blood pressure 160/110
mmHg.
• 24 hour urine is collected in acid container. Urine is
tested for catecholamines.
LC-electrochemical detector results

1. Increased catecholamines.
2. Disproportinate
increase in epinephrine.

Diagnosis:
pheochromocytoma

standard patient sample

 Alpha Fetoprotein

• Hepatocellular carcinoma
• Germ Cell Tumors
– Classifying and staging with hCG
• Nonseminomas: both AFP & hCG elevated (90%)
• Seminomas: AFP not elevated, hCG elevated 30%
• AFP level not directly related to tumor size
• Elevated in pregnancy, liver disease (hepatitis, cirrhosis,
GI tumors)
• AFP Tumor-specific glycoforms may improve specificity
of AFP for HCC
 AFP and fucosylated AFP
Choi et al. Clin Chim Acta 2012;413:170-4.
 CEA

• CEA 150-300 kDa glycoprotein

• Elevated in smokers and elderly
• Elevated in breast, pancreatic, GI, and lung cancer
– Breast cancer: used for detecting and monitoring
metastatic disease
• Elevated in benign diseases: cirrhosis, emphysema &
rectal polyps
• CEA – Not useful for CRC Screening
• New more specific marker for CRC: TIMP-1 (Tissue
inhibitor of metalloprotease)
 CA 15-3/CA27.29

• High molecular weight glycoprotein (Polymorphic

Epithelial Mucin)
• Breast cancer marker
– Correlate with stage and tumor size
– Prognosis & predict response to chemotherapy
– Detect residual disease following initial therapy
– Detect recurrence, correlates with disease progression or
regression
– NOT sensitive enough for early detection
• Elevated in benign diseases of liver & breast
• Elevated in other cancers: pancreatic, lung, ovarian,
colorectal, & liver
 CA 125

• >200-2000 kDa glycoprotein

• Increased in benign diseases: pregnancy, endometriosis,
ovarian cysts, PID, cirrhosis, hepatitis, pericarditis
• Increased in other cancers: lung, breast, GI, endometrial,
& pancreatic
• Synthesis modified by Taxol
Discordance of tumor marker assays due to
variable glycosylations

No Glycosylation: Glycosylation:
glycosylation no effect Major effect

▓▓▓▓

▓▓▓▓

x
▓▓▓ Glycosylation
 CAP Tumor marker PT survey

Vendor TM-01 TM-02 TM-03

Abbott Arch 167 57 20
Beckman Coulter 82 26 9
Roche 125 41 15
Siemens Centaur 114 37 15
Siemens Immulite 71 24 8
Tosoh 176 58 18
Ortho Vitros 113 34 10
Effect of changing tumor marker assays

Method A Method B
Tumor marker level

x Disease progression?
Change therapy?
Analytical difference?
x x
x
x x

0 10 20 30 40 50

Time, weeks
Solutions to discordant tumor marker assay
results
New sample arrives:
• Never change assays (Memorial Sloan Kettering has
assays dating to the 1970s). Not usually practical.
• Perform testing of new sample by both technologies.
Old technology may not be still available or is costly.
• Bank samples for 1-2 years in anticipation of change.
With request of a new sample, retrieve old sample
and “rebaseline” using new assay.
Effect of changing tumor marker assays
Result of old
sample on
Tumor marker level

Method A new method Method B

x x
No change in disease

x x
x
x
x

0 10 20 30 40 50

Time, weeks
 Case report: breast cancer
Ishikawa et al. J Thor Dis 2012;4:epub

• 35 y F admitted for DIC. CEA and CA15-3 increased.

• MRI, mammogram not definitive. Core needle biopsy
revealed invasive ductal carcinoma of the breast. ER,
PR, her-2/neu were negative.
• Started on paclitaxel dropping CA 15-3, but CEA began
to rise. Developed respiratory dyspnea. Switched to
epirubicin/cyclophosphamide reducing CEA and CA15-3.
• Developed jaundice and liver disease. Vinorelbine was
selected improving LFTs.
• Rising CEA/CA15-3 with recurrence of dyspnea. Return
to epirubicin and added capecitabine. Patient expired.
Case reports: breast cancer
 Cytokeratin fragment 21-1

• Cytokeratins are intermediate filament structural proteins

found in cytoskeleton of epithelial cells.
• Increased CYFRA 21-1 seen in all histologic types of
lung cancer but especially non-small cell lung cancer.
• CYFRA 21-1 is used for diagnosis, prognosis, and
monitoring after chemotherapy.
• May be increased in benign respiratory disease,
urological, gastrointestinal and gynecological cancers.
Thyroglobulin
 Thyroglobulin as a tumor marker

Monitoring of the recurrence or metastasis of

differentiated thyroid cancer

Differentiated

Papillary cancer Follicular cancer Anaplastic cancer

 Thyroglobulin testing strategies

Anti-Thyroglobulin Ab

Immunoassay LC/MS/MS
 Prostate specific antigen

• PSA Forms/Measurements:
– 55-95% PSA complexed with antichymotrypsin (PSA-
ACT)
– 5-45% free PSA (fPSA)
– Total PSA = fPSA + PSA-ACT
• Total PSA ranges:
– 0-4 ng/mL = Low risk of PCA (22% positive)
– 4-10 ng/mL = diagnostic gray zone (PCA & BPH)
– >10 ng/mL = 40-50% with PCA
 Prostate specific antigen

• Enhancing Differential Diagnosis PCA

– PSA velocity – increases over time
– % fPSA
– PSA density – tPSA/prostatic volume
– Age-race- adjusted reference ranges
 Free PSA (fPSA)

• Unbound portion of PSA is inversely related to

probability of prostatic carcinoma
• Differentiation from carcinoma and BPH
– When the total PSA is between 4 and 10 ng/mL:
%Free PSA Probability of carcinoma
0 - 10 56%
10 - 15 28%
15 - 20 20%
20 - 25 16%
> 25
Prostate specific antigen clinical applications

• Early detection in conjunction with DRE

PSA >10 ng/mL with +DRE = Biopsy
PSA 4-10 ng/mL and –DRE = Biopsy
• Determine success of radical prostatectomy
• Recurrence following treatment
• Monitoring hormonal treatment
Challenges for PSA screening
Schroeder et al. NEJM 2009;360:1320-8

OR for prostate death: 0.80 (0.65-0.98)

1410 screened, 48 treated to prevent
1 death
 Economic model: quality-adjusted life years

Intervention Disease QALY range

Others
Mammography screening breast cancer 10,000-25,000
Medications hypertension 10,000-60,000
Implantable defribrillators AMI & HF 30,000-70,000

PSA screening prostate cancer $15,000 age 50-59 y

$20,000 for 60-69 y
$65,000 for 70-79 y
Cutoff: $50,000 in the US
 Genetic tumor markers and disease

• Oncogenes: • Tumor Suppressors:

– N-ras: leukemia
– K-ras: colon/ gastric
– C-erB-2: Breast/gastric
– N-myc: Breast/Neuro
– c-abl/bcr: CML
– bcl-2: leukemia/lymp
– HER-2/INT2/ATM/
H-ras: Breast
– MCC: colon
– p53: Breast/colon/lung
– RB1: Retinoblastoma
– WT1& 2: Renal
– BRCA1& 2: Breast/
pancreatic/Ovarian
– BRCA1:prostate/stom.
– APC: Colorectal
– MTS1: Melanoma
– DCC: colon/gastric
 Estrogen and progesterone receptors

• ER pos. have more favorable prognosis within first 5 y after

diagnosis
• Hormone therapy blocks binding of estrogen to estrogen
receptors:
– Block receptor using tamoxifen or aromatase inhibitors
– 60% of patients with primary tumors with ER/PR
respond to hormone therapy
• ER/PR measured in tumor tissue by immunohistochemistry
or ELISA (tumor tissues)
 HER-2/neu (c-erbB-2)

• 185 kDa tyrosine kinase growth factor receptor

• Gene amplification/overexpession occurs in 30%
patients & correlates with aggressive disease &
shortened survival
• Moderate negative predictive factor for response
to endocrine therapy or alkylating agents
• Strong predictive factor for response to
trastuzumab (Herceptin)
• Methods approved by FDA: FISH and IHC
Immunohistochemistry for her-2/neu

Negative 3+
Fluorescence in situ hybridization testing
Immunohistochemistry vs. FISH for her-
2/neu testing
Breast cancer survival with herceptin
Kostler et al. Br Cancer J 2003; 89, 983–991

Her-2/neu pos

Her-2/neu neg
ER/PR and her-2/neu status and survival
Onitilo et al. Clin Med Res 2009;7:4-13.
 Hypermethylation of estrogen receptors
van Hoesel et al. Breast Cancer Res Treat 2012;131:859-
69.

ER+  ER- is caused

by hypermethylation
 worse outcomes.

Hypermethylation is
reversible, treatment with
inhibitors controlling
epigenetic modifications
 Other companion diagnostic tests
Barrett et al. Clin Chem 2013;59:198-201.

Biomarker Drug Cancer

Her2.neu Trastuzumab Breast ca.
KRAS Cetuximab, Colorectal
panitumumab
BRAF Vemurafenib Melanoma
ALK Fusion Crizotinib Non-small cell lung ca.
EGFR Gefitinib, erlotinib Non-small cell lung ca.
BCL-ABL translocation Imatinib, dasatinib, Chronic myeloid
nilotinib leukemia
 RT-PCR for circulating tumor cells

• Prostate Cancer
– PSA, PSMA
• Breast Cancer
– Cytokeratin 19, CEA, MUC1, hMAM
• Melanoma
– Tyrosinase, MART1, MAGE3, GAGE
Mechanism for circulating tumor cells

Metastatic Cascade
Cells grow as benign tumor
Cells break through the
basement membrane
Travel through the blood
Adhere to capillary wall
Escape from blood vessel
(extravasation)
Proliferate to form metastases
CTCs for metastatic breast cancer
Bidard et al. Breast Cancer Res 2012;14:R29
 mRNA Microarrays

• Large mRNA and DNA arrays (Affymetrix, Illumina)

enable unfocused genomic signature analysis.
• Oncotype DX and Mamaprint enable prediction of
therapeutic success in breast cancer.
• Tumor of Origin enables identification of the tissue
origin of metastasis.
Microarray schematic
 Comparative genomic hybridization

• A method of comparing differences in DNA copy

number between tests (e.g. tumor) and
reference samples
• Can use paraffin-embedded tissues
• Good method for identifying gene amplifications
or deletions by scanning the whole genome.
Comparative genomic hybridization
Nature Reviews Cancer 2001;1:151-157
CGH array in inflammatory breast cancer (IBC)
Bekhouche et al. Plos One 2011;6(2):e16950

Inflammatory breast cancer is more lethal due to high metastatic potential

 Expression microarray extraction from tumors
mRNA
sample 1
sample 2
(tumor
(reference)
tissue) cDNA cRNA
cRNA

Cy3-dUTP
Cy5-dUTP green fluorescent
red fluorescent

sample of interest
reverse transcriptase,
compared to
T7 RNA polymerase
standard reference
Microarray results
 Detecting aggressive prostate cancer
Liong et al. PLoS One 2012;7:e45802

• Used Affymetrix gene chip on 255

aggressive vs. 164 non aggressive
prostate cancer patients.
• Developed a 7-member gene panel.
 Oncotype Dx
Paik et al. N Engl J Med. 2004;351: 2817-2826

16 Cancer and 5 Reference Genes

PROLIFERATION ESTROGEN HER2

Ki-67 ER GRB7
STK15 PR HER2
Survivin Bcl2
Cyclin B1 SCUBE2
MYBL2 REFERENCE
Beta-actin
GSTM1 BAG1 GAPDH
INVASION
RPLPO
Stromelysin 3 CD68 GUS
Cathepsin L2 TFRC
 Oncotype Dx
Paik et al. N Engl J Med. 2004;351: 2817-2826

Calculation of the Recurrence Score Result

Coefficient x Expression Level
RS = + 0.47 x HER2 Group Score
- 0.34 x ER Group Score
+ 1.04 x Proliferation Group Score
+ 0.10 x Invasion Group Score
+ 0.05 x CD68
- 0.08 x GSTM1
- 0.07 x BAG1 Category RS (0-100)
Low risk RS <18
Int risk RS ≥18 and <31
High risk RS ≥31
 Oncotype Dx
Paik et al. N Engl J Med. 2004;351: 2817-2826
Stage I-II, node negative, ER+ patients only.
 Oncotype Dx
Paik et al. N Engl J Med. 2004;351: 2817-2826
 Microarray test for tumor of origin
indications over biopsy
• The cancer is found in an unexpected location or multiple
locations, indicating metastatic disease
• Tumor is poorly differentiated or undifferentiated
• Unresolved differential diagnosis of ≥2 cancer types
• The patient has a history of multiple cancers
• IHC are inconclusive or conflicting
• The specimen is small, constraining the diagnostic work up
• Clinical history and histology differ on the dx
• There is an atypical distribution of metastases
• The diagnosis is questioned when the pt fails to respond to tx
Tumor of origin test result
Hereditary cancer genomics
 Cancer genomics examples
Cancer Associated gene Inheritance mode
Breast and ovarian cancer BRCA1, BRCA2 Dominant
Wilms’ tumor WT1 Dominant
Familial retinoblastoma RB1 Dominant
Huntington’s disease Huntingtin Dominant
Hereditary colorectal cancer MLH1, MSH2,6, PMS1,2 Recessive
Skin cancer Xeroderma pigmentosum Recessive
XPB, XPD, XPA
 Self assessment questions

Which technique is most useful for detecting gene

duplications and deletions?
A. Immunohistochemistry
B. Comparative genomic hybridization
C. Fluorescence in situ hybridization
D. Real-time polymerase chain reaction
E. Chemilluminescence immunoassay

Answer: B. CGH arrays are performed on microchips.

 Self assessment questions

Tumor markers that are glycosylated proteins:

A. Are identical between tumors
B. Can cause falsely high and low results by
immunoassays
C. The extent of glycosylation is indicative of disease
severity
D. Assay inaccuracies can be corrected by
standardization
E. Are detected by genomic microarrays

Answer: B. Variation in tumor marker expression result

in discordance between commercial immunoassays.
 Self assessment questions

High sensitivity PSA assays are useful for:

A. Early detection of disease recurrence after
prostatectomy
B. Differentiation between benign prostatic hypertrophy
and prostate cancer
C. Differentiation between aggressive vs. non-
aggressive disease
D. Improved screening for prostate cancer
E. Selection of hormone vs. chemotherapy

Answer: A. As much as 2 years can be gained in some

studies