Biomarkers in cancer

Biomarkers in Cancer

Priya TiwariMedical Oncology

All India Institute of Medical Sciences

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

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

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

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

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

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

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

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

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

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

Some Tumor Bio Markers

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

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

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

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)

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

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)

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

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

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

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

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

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

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

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

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

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

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)

Cancer MarkerBreast Tissue ER, PgR (some uterine and lung cancers are

weakly positive)

Gross cystic disease proteinColon/intestine Tissue CDX2Lung Tissue TTF1 (also positive in thyroid cancer, but

thyroid also positive for thyroglobulin)

Melanoma Tissue S100, Melan-A, HMB45, MITF

Ovarian WT1Prostate 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

Accepted Biomarkers Useful as Predictive Factors for Treatment in Common Solid Malignancies

Cancer Marker TreatmentBreast ER Endocrine

HER2 Trastuzumab; lapatinibColon KRAS mutations Cetuximab; panitumumabLung EGFR mutations

ALK positive

Tyrosine kinase inhibitors (erlotinib, gefitinib)Crizotinib

Accepted Biomarkers Useful for Monitoring of Common Solid Malignancies

Cancer Marker Specific SituationBreast CA 15-3, CA 27.29

Circulating Tumor CellsMonitor 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 NoneMelanoma NoneOvarian CA 125 Monitor patients after primary and adjuvant chemotherapy

for relapseMonitor 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 relapseMonitor patients with metastatic disease

Male germ line malignancy

β-hCG; AFP Monitor patients after primary and adjuvant chemotherapy for relapseMonitor patients with metastatic disease

Female choriocarcinoma

β-hCG Monitor patients after primary and adjuvant chemotherapy for relapseMonitor patients with metastatic disease

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

Single cancer specific

biomarker

Combination of multiple markers

Lack of SensitivityLack of organ specificityLack of disease specificity

?Impact over decision making

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

Newer Advancements in the Field of Biomarkers

ConclusionsFive-gene signature is closely associated with relapse-free and overall survivalamong 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).

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.

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

PROLIFERATIONKi-67

STK15Survivin

Cyclin B1MYBL2

ESTROGENERPR

Bcl2SCUBE2

INVASIONStromelysin 3Cathepsin L2

HER2GRB7HER2

BAG1 GSTM1

REFERENCEBeta-actinGAPDHRPLPO

GUSTFRC

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

Oncotype Dx: Recurrence Score40

35

30

25

20

15

10

5

00 5 10 15 20 25 30 35 40 45 50

Recurrence Score

Rat

e of

Dis

tant

Rec

urre

nce

at 1

0 ye

ars

95% C.I.Recurrence Rate

LowRS < 18Rec. Rate = 6.8%C.I. = 4.0% - 9.6%

IntermediateRS 18 - 31Rec. Rate = 14.3%C.I. = 8.3% - 20.3%

HighRS 31Rec. Rate = 30.5%C.I. = 23.6% - 37.4%

Paik S. et al. N Engl J Med 2004;351:2817-26

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.

MiRNA in Cancer Diagnosis and Prognosis

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

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.

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.

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.

microRNAs Tumorigenesis Diagnosis PrognosismiR-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

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

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

PrognosisCell 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

Circulating Tumor Cells

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

Thank You

Roche Chip for Cytochrome P450Genes: 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

Traditional vs High-throughput approach