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

x Disease progression? Change therapy? Analytical difference? x x x x x Tumor marker level Tumor

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 Method A new method Method B

x x No change in disease

x x x x x Tumor marker level Tumor

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 – p53: Breast/colon/lung – K-ras: colon/ gastric – RB1: Retinoblastoma – C-erB-2: Breast/gastric – WT1& 2: Renal – N-myc: Breast/Neuro – BRCA1& 2: Breast/ – c-abl/bcr: CML pancreatic/Ovarian – bcl-2: leukemia/lymp – BRCA1:prostate/stom. – HER-2/INT2/ATM/ – APC: Colorectal H-ras: Breast – MTS1: Melanoma – MCC: colon – 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 INVASION GAPDH Stromelysin 3 RPLPO 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