Biomarkers have a diversified role in diagnosis, prognostication and risk stratification. This presentation aims to compile the basic information and new literature on various biomarkers pertaining to cancer care.

1. Biomarkers in Cancer
Priya Tiwari
Medical Oncology
All India Institute of Medical
Sciences

2. Definition
• “A biological molecule found in blood, other body fluids, or tissues
that is a sign of a normal or abnormal process, or of a condition or
disease
• Tumor markers: types of biomarkers that can be found in the body
when cancer is present
• Require special assay that is beyond routine clinical, radiographic,
or pathologic examination
“Biomarker"NCI Dictionary of Cancer Terms. National Cancer Institute

3. Types
Prognostic
biomarker
Predictive
biomarker
Disease
related
Drug related
Course of disease irrespective
of treatment used e.g
-Presence of involved local-
regional lymph nodes
Response to a particular treatment

4. 4
In colon cancer KRAS mutation determines response
to EGFR therapy
Mutant KRAS
+EGFR
-EGFR
Wild type KRAS
+EGFR
-EGFR
Amado et al. J Clin Oncol; 26:1626-1634 2008
KRAS mut PIK3CA mut
BRAF mut

5. Types
Prognostic
biomarker
Predictive
biomarker
Disease
related
Drug related
Course of disease irrespective
of treatment used e.g
-Presence of involved local-
regional lymph nodes
Response to a particular treatment e.g.
-KRAS mutations and antiepidermal growth factor
receptor [EGFR] antibody therapies
Many are mixed biomarkers i.e. carry both prognostic and predictive value e.g HER2 neu:
• Amplification or overexpression of HER2 is associated with worse prognosis in
absence of therapy
• HER2 favorable predictive factor for some types of therapy, e.g anthracycline or
taxane-based chemotherapy, and anti-HER2 therapies (trastuzumab and lapatinib)
In NSCLC
• High expression of excision repair cross-complementation gene-1 (ERCC1) associated
with decreased response to platinum-based chemotherapy, but with better overall
prognosis
• Ribonucleotide reductase M1 (RRM1) overexpression correlates with better de
novo prognosis but resistance to gemcitabine
Olaussen KA et al. N Engl J Med 2006;355:983

6. Specimen
• Measured at multiple levels:
– DNA
• Gene mutations, deletions, amplifications, or methylation
– RNA
• Micro RNA,mRNA
– Protein
• Overexpression, under expression, or qualitative abnormalities
– Cells or Tissue
• Presence of cells outside their milieu e.g in circulation
• Demonstration of neovascularization in tissue specimen

7. Clinical Usefulness
• Risk determination
– Adjust risk categorization for individual not affected by disease
– E.g. BRCA 1 & 2 mutation analysis
• Screening
– Prostate specific antigen for carcinoma prostate
• Differential diagnosis
– Analysis of circulating α-fetoprotein (AFP) or β–human chorionic
gonadotropin (β-hCG) in males with poorly differentiated malignancies
of uncertain origin
• Prognosis
– Presence of involved local-regional lymph nodes associated with
subsequent distant recurrence
• Prediction
– Estrogen-receptor content for endocrine therapy
• Monitoring

8. Some General Points About Tumor Biomarkers
• No serum marker in current use is specific for malignancy
• Generally, serum marker levels are rarely elevated in patients with
early malignancy
– With a few exceptions, high levels are usually found only when
patients have advanced disease.
• No cancer marker has absolute organ specificity
– PSA relatively specific for prostate tissue, but not for prostate
cancer

9. General Points…..
• No marker is elevated in 100% of patients with particular malignancy
– Exception: hCG in choriocarcinoma
• Requesting of multiple markers (such as CEA and the CA series of antigens)
in an attempt to identify metastases of unknown primary origin is rarely of use
• Tumour markers assays should not be carried out on biological fluids e.g
(peritoneal fluids, pancreatic juice and ovarian cystic fluids) as reliable
reference ranges currently unavailable

10. General Points…
• Reference ranges for cancer markers are not well defined and are used only
for guidance
• Level below the reference range does not exclude malignancy while
concentrations above the reference range does not necessary mean the
presence of cancer
• Changes in levels over time are likely to be more clinically useful than
absolute levels at one point in time
• As many tumour markers lack agreed International Reference Preparations
(e.g CA125, CA15-3, CA19-9), different assay kits may give different
results for the same sera
• Laboratories carrying out tumour marker tests should state the assay used
on their report form

11. Ideal Tumor Markers
• Be specific to the tumor
• Level should change in response to tumor size
• Abnormal level should be obtained in presence of micrometastases
• Level should not have large fluctuations that are independent of changes in
tumor size
• Levels in healthy individuals are at much lower concentrations than those
found in cancer patients
• Predict recurrences before they are clinically detectable
• Test should be cost effective

12. Some Tumor Bio Markers

13. Alpha Feto Protein
• 70 kDa glycoprotein homologous to albumin
• Forms in serum:
– Exhibits micro heterogeneity due to varying levels of
glycosylation
– AFP produced by malignancies more highly fucosylated than
formed by normal tissues
– Existing assay do not differentiate between various forms
• Half life: 5-7 days

14. Alfa Feto Protein
• Mainly confined to 3 malignancies, i.e.
a. Germ cell tumours (NSGCT) of testis, ovary and other sites
b. Hepatocellular carcinoma (HCC)
c. Hepatoblastoma (in children, extremely rare in adults)
• Benign conditions
– Hepatitis
– Cirrhosis
– Biliary tract obstruction
– Alcoholic liver disease
– Ataxia telangiectasia
– Hereditary tyrosinaemia
Physiological conditions
with elevated levels:
• Pregnancy
• 1st year of life

15. AFP: Clinical Applications
• In combination with hCG, for monitoring patients with NSGCT
• Independent prognostic marker for NSGCT (e.g. of the testis)
• Diagnostic aid for HCC and hepatoblastoma.
– In patients with cirrhosis and a focal lesion > 2 cm with arterial
hypervascularization, an AFP level >200 µg/L is suggestive of HCC,
and AFP>400 µg/L is strongly suggestive of HCC
• Screening for HCC in high risk populations (e.g. in patients with cirrhosis
due to hepatitis B or C)
– 6-monthly AFP measurement and abdominal ultrasound, with AFP>200
µg/L and rising
Not a useful marker for liver metastases

16. CA125
• Protein detected by this antibody is Muc16
• Physiological function: None established
• Malignancies with elevated levels:
– Epithelial ovarian cancer; 80 - 85% of all cases; but increased in only
half of early (stage 1) cancer
– May be elevated in any adenocarcinoma with advanced disease
• Benign conditions with elevated levels:
– Endometriosis
– Acute pancreatitis
– Cirrhosis
– Peritonitis
– Inflammatory pelvic disease
– Presence of ascites (of non-malignant origin)
Physiological conditions with elevated
levels:
• Menstruation & pregnancy (usually
< 100 kU/L)

17. CA-125
• Reference range: 0 - 35 kU/L (most frequently used range)
• Half life : Approx. 5-7 days
• Clinical application:
– Measurement in postmenopausal patients with pelvic masses may help
differentiate malignant from benign lesions
– Rate of decline during initial therapy is an independent prognostic
indicator in ovarian carcinoma
– Monitoring treatment with chemotherapy
– Surveillance following initial treatment
• Unclear impact on survival

18. CA 19-9
• Mucin reacting with monoclonal antibody 111 6 NS 19-9
• Physiological function: Involved in cell adhesion
• Reference range : Very variable, from 0 - 37 kU/L to 0 - 100 kU/L
• Half life in serum: Approx. 1 day (can vary from <1 day to 3 days)

19. CA-19-9
• Malignancies with elevated levels
– Most pancreatic adenocarcinomas
– Approx. 50% of gastric carcinomas
– approx. 30% of colorectal carcinomas.
• Benign conditions with elevated levels
– Acute and chronic pancreatitis
– Hepatocellular jaundice ;Cirrhosis
– Acute cholangitis
• Main clinical applications
– As diagnostic aid for pancreatic carcinoma
• Inadequate sensitivity & specificity limit the use in early diagnosis
– Monitoring treatment of patients with pancreatic adenocarcinoma
– Diagnostic aid in gastric and cholangio carcinomas

20. CA 15-3
• Transmembrane glycoprotein encoded by MUC1 gene
– Defined by reactivity with 2 monoclonal antibodies, i.e., DF3
and 115D8 in sandwich immunoassay
• Physiological function: Involved in cell adhesion & cancer
pathogenesis
• Reference range :0 – 25 to 0 – 40 kU/L
• Half life in serum :Unknown

21. CA15-3
• Malignancies with elevated levels :
– Breast adenocarcinomas, especially with distant metastasis
• Rarely elevated in patients with local breast cancer
• Benign diseases with elevated levels
– Benign liver disease
– Benign breast disease (possibly)
• Main clinical applications:
– Preclinically detecting recurrences in asymptomatic patients with
diagnosed breast cancer
• Controversial
– For monitoring the treatment of patients with advanced breast
cancer

22. CEA
• 200 kDa (approx.) glycoprotein
• Physiological function: Role in cell adhesion & inhibition of
apoptosis
• Reference range: 0 - 3.5 µg/L to 0 - 5.0 µg/L.
• Half life in serum :Approx. 3 days but can vary from 1 to 5 days

23. CEA
• Malignancies with elevated levels
– Elevated in almost any advanced adenocarcinoma, i.e., where distant
metastases present
• Almost never elevated in early malignancy
• Benign diseases with elevated levels:
– Involving liver :Hepatitis, cirrhosis, alcoholic liver disease
– Obstructive jaundice
– Ulcerative colitis, Crohn’s disease
– Pancreatitis
– Bronchitis, emphysema
– Mildly elevated in smokers
• Main clinical applications
– In surveillance following curative resection of colorectal cancer
– In monitoring therapy in advanced colorectal cance

24. Human Chorionic Gonadotropin (hCG)
• Heterodimer composed of 2 glycosolated sub-units (alpha & beta chains)
– Alpha chain is almost identical to alpha chain in TSH, FSH & LH
– Beta chain is distinct from corresponding chains
• Distinctive 24 amino acid carboxy-terminal extension
• Forms in serum: Multiple forms
– Intact 2-chain peptide
– Free alpha and beta chains
– Various degradation products (e.g., beta core fragment)
• Physiological function: to maintain progesterone production by corpus
luteum during early pregnancy
– Can be detected as early as one week after conception

25. hCG
• Malignancies with elevated levels
– Virtually all patients with gestational trophoblastic disease (GTD)
(i.e., complete and partial molar pregnancy, choriocarcinoma and placental
site trophoblastic tumours)
– Non-seminomatous germ cell tumours (NSGCT)
– Seminomatous germ cell tumours of testis (approx. 20%).
• Benign Diseases with elevated levels
– Ectopic pregnancy
– Pituitary adenoma
• Main clinical applications
– For monitoring patients with GTD
– In conjunction with AFP, for determining prognosis and monitoring
patients with NSGCT of testis, ovary and other sites

26. hCG
• Type of sample for assay
– Serum or urine
• Reference range : Serum: 0 - 5 IU/L
• Half life in serum: Approx. 16 - 24 hours; decline may be biphasic
with a second half life of 13 days

27. Prostate Specific Antigen (PSA)
• 28.4 kDa single chain chymotrypsin-like serine protease containing 237
amino acids
• Forms in serum
– PSA complexed with
• A1antichymotrypsin (PSA-ACT) (major)
• A1 -antitrypsin (trace quantity)
• A2 -macroglobulin (undetectable by current immunoassays)
• Non-complexed free form (fPSA) represents 5 - 40% of the “total”
PSA
• Physiological function
– Partially responsible for the liquefaction of semen to promote the
release and motility of spermatozoa

28. PSA…..
• 5-alpha-reductase inhibitors used to treat BPH reduce PSA levels by approx.
50%
• Half life in serum: Approximately 2.5 days after radical prostatectomy; after
radiotherapy may be many months
• Reference range: 0 - 4 µg/L (most frequently used)
Effects of urological manipulations on PSA levels
• DRE: May cause minor increases ; rarely of clinical significance.
• Prostate massage: May cause minor elevations
• TURP: Increases PSA levels significantly. Wait >/=6 weeks before testing
• Needle Biopsy: Increases PSA levels significantly. Wait >/=6 weeks before testing
• Cystoscopy: No change by flexible cystoscopy but rigid cystoscopy may increase levels

29. Prostate Specific Antigen (PSA)
• Main clinical applications
– In combination with digital rectal examination PSA can aid diagnosis
of prostate cancer
– Determining prognosis in patients with prostate cancer
– Surveillance following diagnosis of prostate cancer
– Monitoring therapy in patients with diagnosed prostate cancer
– As screening tool : Controversial
– No significant reduction in mortality from prostate cancer1
– 20% reduction in mortality but at expense of overdiagnosis2
1)Andriole GL.N Engl J Med 2009;360:1310-1319.
2)Schröder FH.N Engl J Med 2009;360:1320-1328
• PSA Density - Normalized to prostate volume
• PSA Velocity - Change in PSA over time (e.g., more than 15% per year)
• Free PSA/Total PSA - lower ratio suggests cancer, since more free PSA from
normal prostate is degraded (< 10% - biopsy)

31. Cancer Marker
Breast Tissue ER, PgR (some uterine and lung cancers are
weakly positive)
Gross cystic disease protein
Colon/intestine Tissue CDX2
Lung Tissue TTF1 (also positive in thyroid cancer, but
thyroid also positive for thyroglobulin)
Melanoma Tissue S100, Melan-A, HMB45, MITF
Ovarian WT1
Prostate Circulating or tissue PSA, urinary PCA3
Male germ cell Tissue or circulating α-fetoprotein, β–human chorionic
gonadotropin (β-hCG)
Tissue PLAP
Accepted Biomarkers Useful for Differential
Diagnosis of Common Solid Malignancies

32. Accepted Biomarkers Useful as Predictive Factors
for Treatment in Common Solid Malignancies
Cancer Marker Treatment
Breast ER Endocrine
HER2 Trastuzumab; lapatinib
Colon KRAS mutations Cetuximab; panitumumab
Lung EGFR mutations
ALK positive
Tyrosine kinase inhibitors
(erlotinib, gefitinib)
Crizotinib

33. Accepted Biomarkers Useful for Monitoring
of Common Solid Malignancies
Cancer Marker Specific Situation
Breast CA 15-3, CA 27.29
Circulating Tumor Cells
Monitor selected patients with metastatic disease
Colon CEA Monitor patients after primary and systemic adjuvant
chemotherapy to detect resectable relapse
Monitor selected patients with metastatic disease
Lung None
Melanoma None
Ovarian CA 125 Monitor patients after primary and adjuvant chemotherapy
for relapse
Monitor patients with metastatic disease
HE-4 Monitor patients with metastatic disease who are CA
125 negative
Prostate PSA Monitor patients after primary and adjuvant chemotherapy
for relapse
Monitor patients with metastatic disease
Male germ line
malignancy
β-hCG; AFP Monitor patients after primary and adjuvant chemotherapy
for relapse
Monitor patients with metastatic disease
Female
choriocarcinoma
β-hCG Monitor patients after primary and adjuvant chemotherapy
for relapse
Monitor patients with metastatic disease

34. Guidelines For Ordering/
Interpreting Tumor Marker Tests
• Never rely on result of single test
• Order every test from the same laboratory
• Consider half-life of the tumor when interpreting result
• Consider how the Tumor Marker is removed or metabolized

35. Single cancer
specific
biomarker
Combination of multiple
markers
Lack of Sensitivity
Lack of organ specificity
Lack of disease specificity
?Impact over decision making

36. OVA1
• First FDA-cleared protein-based in vitro diagnostic multivariate index assay
• Test 5 proteins in blood sample
– β2-microglobulin, transferrin, apolipoprotein A1, transthyretin
– CA125
• Indicate the likelihood of benign or malignant
• OVA1 identified additional patients with potential malignancies
• Help to guide surgical decisions in patients with pelvic masses
Giede KC et al. Gynecol oncol. 205;99:447-461

37. Newer Advancements in the Field of
Biomarkers

38. Conclusions
Five-gene signature is closely associated with relapse-free and overall survival
among patients with NSCLC.
Dual-specificity phosphatase 6 (DUSP6),
monocyte-to-macrophage differentiation associated protein (MMD),
signal transducer and activator of transcription 1 (STAT1),
v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 3 (ERBB3), lymphocyte-
specific protein tyrosine kinase (LCK).

39. Oncotype Dx
• Quantifies the likelihood of disease recurrence in women with early-stage
hormone ER positive only breast cancer
• Development of a high-throughput, real time, RT-PCR method to quantify
gene expression from fixed tumor tissue samples
• Selection of 250 candidate genes
• Testing the relationship between the 250 candidate genes and risk of
recurrence in a series of 447 pts from three clinical studies
Published literature
Genomic databases
DNA array-based experiments
16 cancer-related genes + 5 reference genes → Oncotype DX (recurrence score)
Paik et al. NEJM. 2004.

40. 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
PROLIFERATION
Ki-67
STK15
Survivin
Cyclin B1
MYBL2
ESTROGEN
ER
PR
Bcl2
SCUBE2
INVASION
Stromelysin 3
Cathepsin L2
HER2
GRB7
HER2
BAG1 GSTM1
REFERENCE
Beta-actin
GAPDH
RPLPO
GUS
TFRC
CD68
Paik et al. N Engl J Med. 2004;351:2817-26.
16 cancer genes and 5 reference genes make up the Oncotype DX
gene panel. The expression of these genes is used to calculate the
recurrence score:
Oncotype DX 21-gene recurrence score

41. Oncotype Dx: Recurrence Score
40
35
30
25
20
15
10
5
0
0 5 10 15 20 25 30 35 40 45 50
Recurrence Score
RateofDistantRecurrenceat10years
95% C.I.
Recurrence Rate
Low
RS < 18
Rec. Rate = 6.8%
C.I. = 4.0% - 9.6%
Intermediate
RS 18 - 31
Rec. Rate = 14.3%
C.I. = 8.3% - 20.3%
High
RS  31
Rec. Rate = 30.5%
C.I. = 23.6% - 37.4%
Paik S. et al. N Engl J Med 2004;351:2817-26

42. Oncotype DXTM
– Low RS associated with minimal chemotherapy benefit
– High RS associated with large chemotherapy benefit
– The Oncotype DX Recurrence Score provides precise,
quantitative information for individual patients on prognosis
across and statistically independent of information on patient age,
tumor size, and tumor grade.

43. MiRNA in Cancer Diagnosis and Prognosis

44. MicroRNA Profile in Diagnosis and Prognosis
• miRNAs are small non-coding RNAs which play
key roles in regulating translation & degradation of
mRNAs
• Genetic and epigenetic alteration may affect
miRNA expression, thereby leading to aberrant
target gene(s) expression in cancers
Yanaihara et al, Cancer Cell, 2006:
- miRNA profiles of 104 pairs of primary
lung cancers and corresponding non-
cancerous lung tissues were analyzed by
miRNA microarrays
- High hsa-mir-155 a expression correlated with
poor survival
Yanaihara et al .Cancer Cell. 2006 Mar;9(3):189-98

45. The role of microRNAs in cancer diagnosis
• With the application of in situ RT-PCR, it was shown that the
aberrantly expressed miR-221, miR-301 and miR-376a were
localized to pancreatic cancer cells but not to stroma or normal
acini or ducts.
• Aberrant miRNA expression offered new clues to pancreatic
tumorigenesis and might provide diagnostic biomarkers for
pancreatic cancer.
Lee EJ, et al. Expression profiling identifies microRNA signature in pancreatic cancer.
Int J Cancer 2007, 120:1046-1054.
Cho WC. MicroRNAs: potential biomarkers for cancer diagnosis, prognosis and targets for therapy.
Int J Biochem Cell Biol 2010.
Cho WC. MicroRNAs in cancer - from research to therapy. Biochim Biophys Acta - Rev Cancer
2010;1805(2):209-217.

46. The role of microRNAs in cancer prognosis
• The expression pattern of miRNAs in pancreatic cancer were
compared with those of normal pancreas and chronic
pancreatitis using miRNA microarrays.
• Differentially expressed miRNAs were identified which could
differentiate pancreatic cancer from normal pancreas, chronic
pancreatitis, or both.
• High expression of miR-196a-2 was found to predict poor
survival of more than 24 months.
Bloomston M, et al. MicroRNA expression patterns to differentiate pancreatic
adenocarcinoma from normal pancreas and chronic pancreatitis. JAMA 2007,
297:1901-1908.

47. The role of microRNAs in cancer prognosis
• Expression of let-7 miRNA was frequently reduced in
human lung cancers, and that reduced let-7 miRNA
expression was significantly associated with shorter
postoperative survival.
• Overexpression of let-7 miRNA in A549 lung
adenocarcinoma cell line inhibited lung cancer cell
growth in vitro.
Takamizawa J, et al. Reduced expression of the let-7 microRNAs in human
lung cancers in association with shortened postoperative survival. Cancer Res
2004, 64:3753-3756.

48. microRNAs Tumorigenesis Diagnosis Prognosis
miR-9 Neuroblastoma
miR-10b Breast cancer
miR-15, miR-15a Leukemia, pituitary adenoma
miR-16, miR-16-1 Leukemia, pituitary adenoma
miR-17-5p, miR-17-92 Lung cancer, lymphoma
miR-20a Lymphoma, lung cancer
miR-21 Breast cancer, cholangiocarcinoma, head & neck
cancer, leukemia
Pancreatic
cancer
miR-29, miR-29b Leukemia, cholangiocarcinoma
miR-31 Colorectal cancer
miR-34a Pancreatic cancer Neuroblastoma
miR-96 Colorectal cancer
miR-98 Head & neck cancer
miR-103 Pancreatic cancer
miR-107 Leukemia, pancreatic cancer
miR-125a, miR-125b Neuroblastoma, breast cancer
miR-128 Glioblastoma
miR-133b Colorectal cancer
miR-135b Colorectal cancer
miR-143 Colon cancer
miR-145 Breast cancer, colorectal cancer
miR-146 Thyroid carcinoma

49. Markers of Pharmacogenomics
• Difference due to :
– Inherited, germ-line differences in genes either responsible for
• Metabolism of drugs
• Target of drugs
– Play important role in assessing benefits & risks for specific
therapeutic strategies

50. Some Examples
Drug Enzyme /genes Effect
5 fluorouracil/capecitabine dihydropyrimidine
dehydrogenase (DPD)
Increased side-effects if
defective enzymes
6-mercaptopurine, 6-
thioguanine, azathioprine
Thiopurine
methyletransferase (TPMT)
Increased side-effects if
defective enzymes
Tamoxifen CYP2D6 Lack of efficacy
Irinotecan UGT1A1 Increased side-effects if
defective enzymes
Gemcitabine, Ara-C NT5C3, FKBP5 (genes) Increased expression
associated with better
response
Li et.al. Cancer Research 2008; 68: (17). Sept. 1, 2008

51. Prognosis
Cell Search
• To detect circulating tumor cells (CTC) in blood
– Nucleated cells ≥ 4 µm in diameter
• Captured from bloodstream using antibodies against EpCAM (epithelial cell
adhesion molecule)
• Before start of chemotherapy for CRPC, detection of ≥ 5 CTCs associated
with inferior OS
• Drop in CTCs to <5 on chemotherapy associated with improvement in OS
• Limitation: lack of detection of CTCs in many men with progressive,
metastatic CRPC
De Bono JS,et al. Clin Cancer Res October 1, 2008:14; 6302

52. Circulating Tumor Cells

53. Conclusion
• Marked development in the field of cancer biomarker
• Incorporation of technologies eg. Proteomics, genomics and
metabolomics to search and validate newer biomarkers
• However , a tumor marker which reliably separates normal from
abnormal and can be detected even in early stages is still missing
• Pharmacogenomics: potential tool for individualized therapy

54. Thank You

55. Roche Chip for Cytochrome P450
Genes: CYPC19 and CYP2D6
Xie and Frueh, , Personalized Medicine 2005, 2, 325-337
• Comprehensive detection of gene variations
• Genotyping of two Cytochrome P450 genes
& provides predictive phenotype of
associated enzymatic activities, using DNA
purified from human blood
• Assay distinguishes 29 known
polymorphisms in CYP2D6 gene, including
gene duplication & gene deletion, & two
major polymorphisms in CYP2C19 gene
• Aids in treatment choice & individualizing
treatment dose

56. Traditional vs High-throughput approach