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TUMOR MARKERS

TUMOR MARKERS. Michaela Králíková Department of Biochemistry Faculty of Medicine Masaryk University. USE OF TUMOR MARKERS. Screening: calcitonin in families with MEN syndrome, AFP in patients with liver cirrhosis, PSA in men > 50 years Dg and diff. dg in symptomatic individuals

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TUMOR MARKERS

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  1. TUMOR MARKERS Michaela Králíková Department of Biochemistry Faculty of Medicine Masaryk University

  2. USE OF TUMOR MARKERS • Screening:calcitoninin families with MEN syndrome, AFPin patients with liver cirrhosis, PSA in men >50 years • Dg and diff. dgin symptomatic individuals • Clinical staging of cancer, is aided by quantitation of the marker, i. e. the serum level of the marker reflects the number of cancer cells present in the body • Monitoringof the disease and estimation of tumor value • Prognostic indicatorof disease progression and patient survival • Detection of cancer recurrence, permits early treatment or a change in therapy • Monitoring of responses to therapy

  3. Tu markerskinetics • Doublingtime = time to double its (serum/plasma) level. Theshorter, the more aggressive Tu growth. • biologicalhalf-life:

  4. CLASSIFICATION OF TUMOR MARKERS • According to proof: humoral, cellular • According to chemicalstructure(glykoproteins, glykolipids, polypeptides, imunoglobulins, polyamines) • According to visceral specificity • According to physiologicalfunction(oncofetalantigens, oncoplacentalantigens, enzymes, hormones, serumproteins, receptors and others)

  5. Visceral specificity • high:calcitonin - medullary carcinoma of the thyroid PSA - prostate cancer NSE - small cell lung cancer hCG - germ-cell tumors AFP- hepatocellularand germ-cell carcinoma • moderate:CA 19-9 - pancreatic cancer CA 125 - ovarian cancer CA 15-3 - breast cancer • low: CEA TPA

  6. Oncofetalantigens • substancesproducedduringfetallife(presentin highconcentrations in the sera offetuses, decrease to lowlevelsordisappearafterthebirth) • reappear in patientswithcancer • Theirproductiondemonstratesthatcertaingenes are reactivated as a resultofthemalignanttransformationofthe cell. SCC(squamous cell carcinoma) MCA (mucinouscarcinoma antigen) MSA (mammaryserum antigen) TATI (tumor associated trypsin inhibitor) • CEA • CA (carbohydrateantigens) • AFP

  7. CEA (carcinoembryonic antigen) • familyofrelatedoncofetal cell-surfaceglycoproteins • nonspecific • ↑:liver cirrhosis, pulmonaryemphysema, benignbreastcystsdisease, ulcerativecolitis, rectalpolypscolorectal, lung, ovarial, pancreatic, gastric and bileducts Ca • markerforcolorectaland breastcarcinoma, pancreatic, gastric and bileducts Ca • cutoffvalue< 5.0 ng/ml

  8. CEA – 1st choise marker of colorectal Ca (CRCA) • One of the most common malignancies in both sexes in economically developed countries • Incidence has increased more than 3 times during last 30 years. • Prevalence is increasing annually by 2−3%. • CR – newly diagnosed around 8 000 patients per year and about half of them die from CRCA. • CR – 3rd most common malignancy in ♂, 4th in ♀ • 25 % is diagnosed metastasized! 2017 data.

  9. Colorectal carcinoma • possibilities of prevention : Secondary Broadcastscreeningfrom 1.7.2000 (CR)-cyclicfecal occult blood testing in asymptomatic individuals from age 50 or screeningcolonoscopyfromage 55. Primary Lifestyle, nutrition

  10. CRCA – other possibilities of detection at an early stage • detectionof earlyadenomalesions or initial phasesof CRCA: • detectionofmutations (PCR, DNA biochips) in faeces and blood: • Detectionofnewbiomarkers: Point mutationsKRAS, APC; mutationsofinstabilitymarkersBAT 26, P53; DNA integrity test In faeces: CNRIP1, INA, MAL, SNCA, SPG20 In blood: matrix metaloproteinase 2 (MMP 2), tissue inhibitor ofmetaloproteinase2 (TIMP 2) Antibodiesagainst RPH3AL (rabphilin 3A-like protein) Monitoring ofserumcytokinesor HLA-G

  11. CA (carboydrateantigens) CA 19-9 CA 15-3 high-molecular-weight mucins or blood group antigens on the tumor cell surfaces or secreted by the tumor cells CA 72-4 CA 19-9, CA 125 CA 19-9, CA 72-4 CA 125

  12. CA 72-4 (carbohydrate antigen 72-4) • glycoproteinproduced by oesophageal, gastric and pancreaticepithelium • in adults↑:liver diseases, acutepancreatitis, gastriculcer, inflammationsof GITCa ofstomach, colon, uterus, lung(NSCLC) • markerformonitoring ofgastric Ca (1st choicemarker), pancreatic, oesophageal and ovarianCa • cutoff≤ 7 IU/ml

  13. CA 19-9 (carbohydrate antigen 19-9) • glycoproteinoffetal GIT, pancreas and liver epithelium; in adultsitisproduced by GIT and bronchialepithelium. • markerforpancreatic, colorectal and gastriccarcinoma • cutoffvalue≤ 40 IU/ml

  14. CA 19-9 • Sensitivity in selectedTu(Klinická biochemie a metabolismus, 2009)

  15. CA 125 (carbohydrate antigen 125) • glycoproteinofairways and digestive tractepitheliumofbothfetuses and adults • ↑:*ovarial, colorectal Ca *endometrial, breast, pancreatic, liver and pulmonaryCa * pregnancy, breastmilk* benigndiseasesofovaries and endometrium, hepatitis, icterus, pancreatitis • markerfordg and monitoring oftherapyof non-mucinousovarianCa; additionalmarkerforpancreaticand colorectalCa • cutoff≤ 35 IU/ml

  16. CA 15-3 (carbohydrate antigen 15-3) • glycoprotein of fetal bronchial and hepatic cells, adult mammary cells • in adults ↑: pregnancy rheumatic dis., chronic dis. of liver, stomach, pancreas, ovaries, uterus, prostatic gland, AIDS Ca of organs mentioned above • marker for breast Ca monitoring • cut off ≤ 35 IU/ml

  17. CA 15-3 and CEA – 1st choicemarkersforbreastcarcinoma • The 2nd most commonCa in females • Incidence – 1 million of womanworldwide • 90 - 95% sporadic • 5 – 10% inherited - BCRA1 and2 genemutations – possibility of DNA testing from peripheral lymphocytes • The lifetime risk of developing cancer for BRCA1/2 is 87%, in women without mutation 8-10%. • Secondaryprevention –mammography orultrasoundexamination from 45 years of age

  18. AFP (a1-fetoprotein) • glycoproteinsynthesized in largequantities by thefetalyolksac and liver • oneofthe major proteins in thefetalcirculation • in adults AFP /S ↑: pregnancy liver diseases • markerforhepatocellular and germ-cell carcinoma • cutoffvalue < 10mg/l

  19. AFP (a1-fetoprotein) • Sensitivity in selected Tu(Klinická biochemie a metabolismus, 2009)

  20. Oncoplacentalantigens • Substancesproduced by thetrophoblasticcellsofthe placenta in bothpregnancy and pathologicalconditions and also by germinativetumors as a markofmalignantdedifferentiation • ↑ levels show evidence of ↑ malignancy and metastaticpotencyofthegiven tumor SP-1 • hCG

  21. hCG (humanchorionic gonadotropin) • glycoproteinsecreted by thesyncytiotrophoblasticcellsofthe placenta • consistsoftwosubunits:a-subunit (common to severalotherhormones, e. g. FSH, LH or TSH)b-subunit(unique to hCG) • ↑:pregnantwomenhydatidiformmole • markerfortumorsof placenta (trophoblastictumors, particularlychoriocarcinoma), and germ-cell tumorsofthetestis and ovary • cutoffvalue < 2.00 IU/l males, < 10.00 IU/l females(bhCG)

  22. Enzymes NSE (neuron specificenolase) TK (thymidinkinase) LD kathepsins • present in much higherconcentrationsinsidecells • releasedintocirculation as theresultof tumor necrosisor a change in themembrane permeability ofthecancercells→ • elevated enzyme levelsmaysignalthe presence ofmalignancy but usually are not specificenough to identify a cancer type or organ involvement • PSA • ALP

  23. Prostate carcinoma • 2nd most common malignancy in men, CR • 7 049 new cases in 2015 (136 new cases/ 100 000 males) • Risk factors: age, life style, genetic factors Year

  24. Epidemiology of prostate cancer

  25. PSA (prostate-specific antigen) • glykoprotein protease (237 AA, Mr = 33 000) produced exlusively by the epitelial cells of the prostate gland, secreted into seminal fluid (liquefaction). • Produced as inactive proPSA → PSA. • In serum, it occurs as free fPSA anda1-antichymotrypsinora2-macroglobulinbound (55-95%). • ↑: benign prostatic hyperplasia BPH, prostate infammation, urological manipulations • marker for screening (men > 50y, urinating difficulties),dg and monitoring of course and treatment of prostate cancer • cut off value <4.0 µg/l(= ng/ml) (> 50 y), 2.5µg/l(< 50 y, see more in age specific levels)

  26. Increased levels of total PSA in plasma / serum • agespecificlevels: cutoff 40-49 y. 2,5 ng/ml, 50-59 y. 3,5 ng/ml, 60-69 y. 4,5 ng/ml, 70 and more y.6,5 ng/ml • tPSA> 10 ng/ml: suspiciousPCa, weperformanotherexaminations • tPSA4 – 10 ng/ml: PCax BHP???, weperformanotherexaminations

  27. Derived parameters • index f/t PSA – free/total PSA:fPSA<15%: probablePCa, fPSA> 20% probablebenigncondition • tPSAD (tPSAdensity): • ratio [tPSA]/UTSprostatevolume in cm3 • adjustmentof BPH and PCa: cutoff0.15 ng/ml • PSAV (tPSAvelocity): • increaseof[tPSA] / year • healthy0.04 ng/ml/y, BPH 0.07-0.27 ng/ml/y, PCa≥ 0.75 ng/ml/y • tPSAdoublingtime: • time to double [tPSA] • tPSA-TZ: • [tPSA] / transitionzonevolume

  28. Other derived parameters

  29. Other causes of PSA increase in blood • Otherprostatediseases:benignprostatehyperplasia, prostaticinflammation • Mechanicalstimulation(fPSAis more susceptible): biopsy, cystoscopy, catetrization, per rectumexamination • Ejaculation • PSA is a prostate-specific biochemical marker but is not specific for cancer.

  30. ALP (alkalinephosphatase) • Zn2+glycoprotein, in alkalineenvironment (pH= 8-10) itcatalysesthehydrolysisof H3PO4monoesters and transphosphorylation • bone isoenzyme (b-ALP) • ↑: osteoSa, bone metastasesotherbone affections; growth • liver isoenzyme (l-ALP) • ↑:liver metastasesotherliver diseases • ref.values : adults 0.5-2.15 mkat/l, 1 month - 15 years 1.35-7.5 mkat/l, newborns1.2-6.3 mkat/l

  31. Hormones ACTH ADH • Theproductionofhormones in cancerinvolvestwoseparateroutes: • theendocrinetissuethatnormallyproducesthegiven hormone canproduceitsexcessamounts • ectopic syndrome - hormone produced by a distantnonendocrinetissuethatnormallydoes not producethishormone (forinstance: ACTH normallyproduced by thepituitarygland,ectopicalyproduced by thelungsmallcells) • elevationof a hormone is not specific← itmaybeproduced by a variety ofcancers • prolactin • calcitonin • PTH 1. 2.

  32. Serumproteins producedeither by tumor cellsor by anorganism in the presence of tumor ferritin β2-microglobulin • paraproteins

  33. Monoclonalimmunoglobulins(paraproteins) • thefirstdescribed tumor markersproduced by neoplastic plasma cells in monoclonalgammopathies. In serum, wecanidentifywholeIg, heavychains (IgG, M, A; D, E) andk, l lightchains (Bence Jones proteins) -these are smallenough (22 kD) to passthroughthekidneyintothe urine→prerenal „over-flow“proteinuria. • ↑: multiplemyeloma and othermonoclonalgammapathies, lymphomas and leukemias, osteogenicsarcoma, bone metastases • markerformultiplemyeloma and othermonoclonalgammapathies • ref. values: FLC (free lightchains)/S:k = 3.3-19.4 mg/l,l = 5.7-26.3 mg/l, indexk/l= 0.26-1.65;polyclonal FLC/U = 1-10 mg/24h;k/U = 1.25-5.5 mg/l,l/U= 0.51-3.2 mg/l, indexk/l= 0.82-3.0; paraprotein/U - obsolete

  34. Receptors Cellular (tissue) markersused in hormone-producingtumors Growthfactorsreceptors (HER1, HER2/neu) DNA aneuploidy • Estrogen rec. • Progesterone rec. Themainusage: breast Ca, colorectal Ca; brain tumors

  35. Estrogen and progesterone receptors • The most important prognostic markers for breast Ca;detected in tumor tissue. • positivity = ↑ cell diferentiation, ↓ invasivity, better prognosis; = antiestrogen therapy indication • immunohistochemical determination: positivity % value • ELISA: cut off < 15 fmol/mg of protein

  36. Growthfactorsreceptors • Transmembrane receptors with tyrosinkinase activity – phosphorylation of Tyr fosforylují tyr residues of protein substrates • The binding of substrate to the extracellular domain causes a conformational change of the receptor,its autophosphorylation and activation of downstream signaling pathways → influence of cel. proliferation, inhibition of apoptosis • HER1 (EGFR), HER/2neu, HER3, HER4

  37. Growthfactorsreceptors * Nomenclature: HER 2 in humans, neu in rodents

  38. Breast carcinoma markers - summary • Basic markers: CEA and CA 15-3 • Receptor markers: • Growth factors receptors • Estrogene receptor • Progesterone receptor • Markers of metastasis and proliferation - see further

  39. Other tumor markers tissues - producedsubstances, whichwecannotclasswiththepreviouslymentionedgroups Chromogranin A Neuropeptide Y S-100 b 5-hydroxyindolacetic acid • TPA, TPS • Mesotelin

  40. TPA (tissue polypeptide antigen) • non-specificcytokeratinsfragmentsproduced by bothnormal and tumor cells • ↑levelsseen in increased cell proliferation→itsestimationisusefulfor monitoring ofthedisease • ↑:liver dis., DM, rheumatoid dis. breastand GIT tumors • markerforurinarybladdercarcinoma • cutoffvalue≤ 140 IU/l

  41. Tumor markers – lungs, bronchi, trachea Non-parvicellular Ca (NSCLC) CEA CYFRA 21-1 Parvicellular Ca (SCLC) NSE CYFRA 21 -1

  42. CYFRA 21-1 (cytokeratin fragment) • Cytokeratin 19 fragment present in lung, uterine and GIT cells. Markerofdegradationofmalignanttissues and necrosis. • ↑:cirrhosis, asthma, respiratoryinfections, renalfailure • markerforcervical and pulmonary (NSCLC) carcinoma • cutoffvalue≤ 3.3 mg/l

  43. NSE (neuron-specific enolase) • enolase - enzyme ofglycolysis(2-phosphoglycerate → phosphoenolpyruvate) • NSE - formofenolasefound in neuronal and neuroendocrinetissues • ↑: lung and liver dis., renalfailure • markerforsmall-cell lungcancer (SCLC), pheochromocytoma, neuroblastoma, medullarycarcinomaofthethyroid, melanoma, and pancreaticendocrinetumors • cutoffvalue < 15 mg/l

  44. Markersfor dg and monitoring of bone metastasis Bone metastases: tumors of lungs, prostate, breast Monoclonal gamapathies New bone formationmarkersUsage: monitoring the effect of treatment on osteoblastic metastases Bone resorption markers Usage: dg bone mass distribution of solid tumors (PCa), monitoring the effect of antiresorptive treatment

  45. Markers in bone metastases Bone formationmarkers • PINP (N-terminalpropeptideof type I procollagen) tropocollagen → collagenmaturation PINP, PICP • Osteocalcin - serum levels are proportional to its formation in osteoblasts. • Bone ALP – bone izoenzyme of ALP, serum levels are proportional to osteoblastsactivity procollagen

  46. Markers in bone metastases Bone resorptionmarkers • ICTP (C-telopeptideof type I collagen): marker of collagendegradationby action of MMP 9 • CTX-I (b-CTX b-Cross Laps, C-terminaltelopeptideof type I collagen):marker of collagendegradationby action of enzymesfromosteoclasts osteoclast cathepsin K MMP 9 tumor cell collagen I CTX-I ICTP NTX-I

  47. Proliferative antigen Ki-67 • The non-histone nuclear protein expressed during active cell cycle phases (max at the G2 interface and mitosis, is absent in the G0 phase). • It affects the spatial layout of chromatin - gene expression control. • Immunohistochemistry detection in biopsy tissue - Anti-Ki-67 antibody. • Ki-67 expression = proliferative tumor activity. • Proliferative activity in cancer correlates with grade and prognosis. = prognostic marker determined in tumor tissue of solid tumors

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