Download Tumor Markers - American Association for Clinical Chemistry

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
Prostate
Lung
Breast
Colorectal
Urinary system
Skin
Pancreas
Ovarian
Myeloma
Thyroid
Germ Cell
New Cases
218,000
222,500
207,500
149,000
131,500
68,770
43,100
22,000
20,200
44,700
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
CA15-3, BR 27.29
CEA, CA 19-9
CA 72.4, CA 19-9, CEA
NSE, CYFA 21.1
PSA, PAP
CA 125
Calcitonin, thyroglobulin
hCG
CA 19-9, CEA
AFP, CA 19-1
BAP, Osteocalcin, NTx
Catecholamines, metabolites
Fecal occult blood
Cancer
Breast
Colorectal
Gastric
Lung
Prostate
Ovarian
Thyroid
Trophoblastic
Pancreatic
Hepatocellular
Bone
Pheochromocytoma
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:
Major effect
Glycosylation:
no 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
Tumor marker level
Method A
Method B
x Disease progression?
Change therapy?
Analytical difference?
x
0
x
x
x
10
x
20
30
Time, weeks
40
50
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.
Tumor marker level
Effect of changing tumor marker assays
Result of old
sample on
new method
Method A
Method B
x
x
No change in disease
x
x
x
x
x
0
10
20
30
Time, weeks
40
50
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 (PSAACT)
– 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
0 - 10
10 - 15
15 - 20
20 - 25
> 25
Probability of carcinoma
56%
28%
20%
16%
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
Medications
Implantable defribrillators
breast cancer
hypertension
AMI & HF
10,000-25,000
10,000-60,000
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:
–
–
–
–
–
–
–
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
• Tumor Suppressors:
–
–
–
–
–
–
–
–
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 her2/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;131:85969.
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
KRAS
Trastuzumab
Cetuximab,
panitumumab
Vemurafenib
Crizotinib
Gefitinib, erlotinib
Imatinib, dasatinib,
nilotinib
Breast ca.
Colorectal
BRAF
ALK Fusion
EGFR
BCL-ABL translocation
Melanoma
Non-small cell lung ca.
Non-small cell lung ca.
Chronic myeloid
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
sample 1
(tumor
tissue)
Cy5-dUTP
red fluorescent
reverse transcriptase,
T7 RNA polymerase
mRNA
cDNA
cRNA
sample 2
(reference)
cRNA
Cy3-dUTP
green fluorescent
sample of interest
compared to
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
Ki-67
STK15
Survivin
Cyclin B1
MYBL2
INVASION
Stromelysin 3
Cathepsin L2
ESTROGEN
ER
PR
Bcl2
SCUBE2
GSTM1 BAG1
CD68
HER2
GRB7
HER2
REFERENCE
Beta-actin
GAPDH
RPLPO
GUS
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
Category
- 0.07 x BAG1
Low risk
RS (0-100)
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
Recessive
Xeroderma pigmentosum
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. nonaggressive 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