How do we diagnose cancer today?

Transcription

1 Tumor Markers Alan H.B. Wu, Ph.D. Professor, Laboratory Medicine, UCSF Section Chief, Clinical Chemistry, Toxicology, Pharmacogenomics Laboratory, SFGH

2 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

3 How do we diagnose cancer today? Physical Examination Blood tests CT scans Biopsy Human Prostate Cancer Normal Blood Smear Chronic Myeloid Leukemia

4 Death rates for cancer vs. heart disease

5 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

6 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.)

7 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

8 Determine risk (PSA) Roles for tumor markers 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)

9 Tumor markers in routine use Marker CA15-3, BR 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

10 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.

11 LC-electrochemical detector results 1. Increased catecholamines. 2. Disproportinate increase in epinephrine. Diagnosis: pheochromocytoma standard patient sample

12 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

13 AFP and fucosylated AFP Choi et al. Clin Chim Acta 2012;413:170-4.

14 CEA CEA 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)

15 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

16 CA 125 > 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

17 Discordance of tumor marker assays due to variable glycosylations No glycosylation Glycosylation: no effect Glycosylation: Major effect x Glycosylation

18 CAP Tumor marker PT survey Vendor TM-01 TM-02 TM-03 Abbott Arch Beckman Coulter Roche Siemens Centaur Siemens Immulite Tosoh Ortho Vitros

19 Effect of changing tumor marker assays Method A Method B Tumor marker level x x x x x x Disease progression? Change therapy? Analytical difference? Time, weeks

20 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.

21 Effect of changing tumor marker assays Tumor marker level x Method A x x Result of old sample on new method x x No change in disease x Method B x Time, weeks

22 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.

23 Case reports: breast cancer

24 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.

25 Thyroglobulin

26 Thyroglobulin as a tumor marker Monitoring of the recurrence or metastasis of differentiated thyroid cancer Differentiated Papillary cancer Follicular cancer Anaplastic cancer

27 Thyroglobulin testing strategies Anti-Thyroglobulin Ab Immunoassay LC/MS/MS

28 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

29 Prostate specific antigen Enhancing Differential Diagnosis PCA PSA velocity increases over time % fpsa PSA density tpsa/prostatic volume Age-race- adjusted reference ranges

30 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 % % % % > 25

31 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

32 Challenges for PSA screening Schroeder et al. NEJM 2009;360: OR for prostate death: 0.80 ( ) 1410 screened, 48 treated to prevent 1 death

33 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 y $20,000 for y $65,000 for y Cutoff: $50,000 in the US

34 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

35 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)

36 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

37 Immunohistochemistry for her-2/neu Negative 3+

38 Fluorescence in situ hybridization testing

39 Immunohistochemistry vs. FISH for her- 2/neu testing

40 Breast cancer survival with herceptin Kostler et al. Br Cancer J 2003; 89, Her-2/neu pos Her-2/neu neg

41 ER/PR and her-2/neu status and survival Onitilo et al. Clin Med Res 2009;7:4-13.

42 Hypermethylation of estrogen receptors van Hoesel et al. Breast Cancer Res Treat 2012;131: ER+ ER- is caused by hypermethylation worse outcomes. Hypermethylation is reversible, treatment with inhibitors controlling epigenetic modifications

43 Other companion diagnostic tests Barrett et al. Clin Chem 2013;59: Biomarker Drug Cancer Her2.neu KRAS BRAF ALK Fusion EGFR BCL-ABL translocation Trastuzumab Cetuximab, panitumumab Vemurafenib Crizotinib Gefitinib, erlotinib Imatinib, dasatinib, nilotinib Breast ca. Colorectal Melanoma Non-small cell lung ca. Non-small cell lung ca. Chronic myeloid leukemia

44 RT-PCR for circulating tumor cells Prostate Cancer PSA, PSMA Breast Cancer Cytokeratin 19, CEA, MUC1, hmam Melanoma Tyrosinase, MART1, MAGE3, GAGE

45 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

46 CTCs for metastatic breast cancer Bidard et al. Breast Cancer Res 2012;14:R29

47 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.

48 Microarray schematic

49 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.

50 Comparative genomic hybridization Nature Reviews Cancer 2001;1:

51 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

52 Expression microarray extraction from tumors sample 1 (tumor tissue) cdna mrna crna crna sample 2 (reference) Cy5-dUTP red fluorescent Cy3-dUTP green fluorescent reverse transcriptase, T7 RNA polymerase sample of interest compared to standard reference

53 Microarray results

54 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.

55 Oncotype Dx Paik et al. N Engl J Med. 2004;351: Cancer and 5 Reference Genes PROLIFERATION Ki-67 STK15 Survivin Cyclin B1 MYBL2 INVASION Stromelysin 3 Cathepsin L2 ESTROGEN ER PR Bcl2 SCUBE2 GSTM1 CD68 BAG1 HER2 GRB7 HER2 REFERENCE Beta-actin GAPDH RPLPO GUS TFRC

56 Oncotype Dx Paik et al. N Engl J Med. 2004;351: Calculation of the Recurrence Score Result RS = Coefficient x Expression Level x HER2 Group Score x ER Group Score x Proliferation Group Score x Invasion Group Score x CD x GSTM x BAG1 Low risk RS <18 Category RS (0-100) Int risk RS 18 and <31 High risk RS 31

57 Oncotype Dx Paik et al. N Engl J Med. 2004;351: Stage I-II, node negative, ER+ patients only.

58 Oncotype Dx Paik et al. N Engl J Med. 2004;351:

59 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

60 Tumor of origin test result

61 Hereditary cancer genomics

62 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

63 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.

64 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.

65 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