Genetic Testing in Medullary Thyroid Carcinoma Presenter-Dr Sunil Malla Bujar Barua Moderator- Prof Gaurav Agarwal 1 Genetic testing in MTC 24/4/2012
Background 1959 Hazard et al first described MTC 1961 John Sipple recognized the association between thyroid cancer & pheochromocytoma 1965 Schimke & Hartman described syndrome of pheochromocytoma & MTC with amyloid stroma 1968 Steiner and colleagues described the association between MTC, pheochromocytoma, and hyperparathyroidism as multiple endocrine neoplasia (MEN), type 2. 1987 Matthew Simpson MEN 2A putative locus 10p12 p11 10q11.2 1993 Mulligan, Donis-Keller Identification of MEN2 gene (RET) in MEN 2/FMTC families Specific RET Proto-oncogene mutations 2 Genetic testing in MTC 24/4/2012
Medullary Thyroid Cancer MTC represents approximately 4% of all thyroid cancers. Sporadic 75% of cases (non-inherited, presenting typically in the fourth decade as a unifocal lesion without associated endocrinopathy. Inherited 25% of cases 95% of MEN2A and MEN 2B cases have germline mutations 88% of familial MTC cases have germline mutations 25% (up to 50%) of sporadic MTC cases have somatic mutations
RET RET is an abbreviation for "rearranged during transfection" DNA sequence originally found to be rearranged within a 3T3 fibroblast cell line following its transfection with DNA taken from human lymphoma cells 4 Genetic testing in MTC 24/4/2012
RET protein Is a trans cell membrane receptor with a extracellular and an intracellular portion. Transmits signals for growth depending upon stimulation by growth factors which attach to the extracellular portion of receptor Has activating and deactivating mutations Plays role in development of - Intestinal neurons -Autonomic nervous system -Normal kidney development -Spermatogenesis 5 Genetic testing in MTC 24/4/2012
Crystallographic structure dimeric tyrosine kinase domain of the human Ret proto-oncogene (Nterminus = blue, C- terminus= red). Knowles PP et al. J. Biol. Chem. 281 (44): 33577 87. 6 Genetic testing in MTC 24/4/2012
Components of the RET Signaling Pathway Co-receptors: GFR 1, 2, 3, & 4 Growth Factor Ligands: GDNF, NRTN, ARTN, PSPN
Cys GDNF GFR 1 Cys Cys Cys Extracellular Domain Cell Membrane P P Intracellular Domain RAS-RAF Pathway PI3 Kinase Pathway
FMTC/MEN2-associated Mutations in the RET Proto-oncogene Cadherin-like domain Codon mutated Exon MEN2 phenotype Frequency of mutation in MEN2A/FMTC or MEN2B Cysteine-rich domain Transmembrane region 609 10 FMTC/MEN2A 611 FMTC/MEN2A 618 FMTC/MEN2A 23 % 620 MEN2A/FMTC 630 11 FMTC 634 MEN2A/FMTC 66 % Tyrosine kinase 1 Tyrosine kinase 2 768 13 FMTC 1% 790 MEN2A/FMTC 791 MEN2A/FMTC 8 % 804 FMTC 1% 844 14 FMTC 1% 883 15 MEN2B 3% 891 FMTC 1% 918 16 MEN2B 95% 9 Genetic testing in MTC 24/4/2012
The Effects of Mutations on RET Function Ligand independent dimerization, activation, and transformation. Constitutive Phosphorylation of intracellular proteins. Overactive Signaling
Cys GFR 1 Cys Cys Arg Extracellular Domain Cell Membrane P P P P Intracellular Domain Over Active Signalling to Cells Uncontrolled Cell Proliferation
Polymerase Chain Reaction 12 Genetic testing in MTC 24/4/2012
Discovery PCR discovery widely credited to Kary Mullis-1983 On a long motorcycle drive Mentally visualized the process Nobel Prize in Chemistry 1993
Properties of DNA polymearse Needs a pre-existing DNA to duplicate Cannot assemble a new strand from components Called template DNA Can only extend an existing piece of DNA Called primers 5 3 3 5
Properties of DNA polymearse DNA polymerase needs Mg ++ as cofactor Each DNA polymerase works best under optimal temperature, ph and salt concentration PCR buffer provides optimal ph and salt condition
Properties of DNA polymearse DNA strands are anti-parallel One strand goes in 5 3 The complementary strand is opposite DNA polymerase always moves in one direction (from 5 3 ) 5 3 3 5
Properties of DNA polymearse DNA polymerase incorporates the four nucleotides (A, T, G, C) to the growing chain dntp follow standard base pairing rule datp datp datp datp datp dgtp dttp dgtp dctp dttp dctp 5 dttp dttp dctp 3 dgtp datp dctp 3 5 dttp datp dctp dttp dgtp dgtp dctp dgtp
Properties of DNA polymearse The newly generated DNA strands serve as template DNA for the next cycle PCR is very sensitive
Setting up a PCR Reaction Add template DNA and primers Add dntps Add DNA polymerase dttp datp datp datp datp datp dgtp dttp dgtp dctp dctp 5 dttp dttp dctp 3 dgtp datp dctp 3 5 dttp datp dctp dttp dgtp dgtp dctp dgtp
Taq DNA polymerase Derived from Thermus aquaticus Heat stable DNA polymerase Ideal temperature 72C
Thermal Cycling A PCR machine controls temperature Typical PCR go through three steps Denaturation Annealing Extension
Denaturation Heating separates the double stranded DNA Denaturation Slow cooling anneals the two strands Renaturation Heat Cool
Annealing Two primers are supplied in molar excess They bind to the complementary region As the DNA cools, they wedge between two template strands Optimal temperature varies based on primer length etc. Typical temperature from 40 to 60 C
Extension DNA polymerase duplicates DNA Optimal temperature 72C
PCR Amplification Exponential Amplification of template DNA
Typical PCR mix In a thin wall Eppendorf tube assemble the following PCR components Template DNA (5-200 ng) 1 mm dntps (200 um final) 10 X PCR buffer 25 mm MgCl2 (1.5 mm final) 20 um forward primer (20 pmoles final) 20 um reverse primer (20 pmoles final) 5 units/ul Taq DNA polymerase (1.5 units) Water Final Volume Amount variable 10 ul 5 ul 3 ul 1 ul 1 ul 0.3 ul Variable 50 ul
What is genetic counseling? Process through which individuals affected by or at risk for a problem which may be genetic or hereditary, are informed of Consequences of disorder Probablity of suffering and transmission to offspring Means of treating or avoiding occurrence of malformation or disease
Key Issues with genetic testing Can we claim confidentiality over our genetic information? What personal consequences does genetic information have? What implications does it have on family members? Who should have access to the information? Employers? Insurance companies? Government? Meet the Gene Machine
Genetic Counselor Health care professional with a master degree in human genetics and counseling. In common disorders- family doctor, pediatrician or pediatrician
Pretest Genetic counseling Implications should be discussed with family members Genetic transmission, probability of inheritance Risk and benefits Potential of genetic discrimination in employment Life and health insurance Privacy issues Potential in genetic testing errors Potentials of future technologies prenatal testing Therapy Role of life long surveillance Psychological and social support mechanisms should be available Written consent Post test counseling 34 Genetic testing in MTC 24/4/2012
Why Screen? 25% of all MTCs are Familial* Up to 7% of persons with apparently sporadic MTC or pheochromocytoma have RET proto-oncogene mutation* May follow diffuse hyperplasia nodular hyperplasia medullary thyroid carcinoma sequence At the stage of palpable nodule- tumor is nearly 1 cm and 50% may have metastasis in neck nodes** *Elisei etal. J Clin Endocrinol Metab 2007 92(12):4725-4729 **Leboulleux etal. Clin Endocrinol 2004; 61:299-310 35 Genetic testing in MTC 24/4/2012
Why Screen? The aim of screening depends on the phenotypic expression MTC - for prophylactic intervention PCC - for early detection and appropriate management HPT - for early detection and appropriate management 36 Genetic testing in MTC 24/4/2012
Whom to screen? Asymptomatic 1 st degree relatives of a RET mutation carrier proband Relatives expressing one or the other components. Two Methods Testing for all RET exons 10,11,13,14,15,16. Focused testing for known RET mutation in the proband Brandi etal. J Clin Endocrinol Metab 2001;86(12):5658-5671 37 Genetic testing in MTC 24/4/2012
How to screen? Genetic Screening RET mutation analysis 38 Genetic testing in MTC 24/4/2012
Genetic screening vs Calcitonin Genetic Testing: Once in a life time Easy to perform Prophylactic Thyroidectomy possible Early detection before biochemical postivity Spared the expense & inconvenience of repeated testing Both prenatal & preimplantation testing available Detect 95% of pts with MEN 2A, MEN 2B & 88% with FMTC False positive rate <1% Basal/Stimulated Calcitonin : Positive test indicates Cancer has already developed Provocative tests Unpleasant & uncomfortable Annual tesing Assess the burden of disease Important in monitoring for residual/recurrent disease after Thyx False positive rate 5-10% Learoyd et al, Arch Surg 1997;132:1022-1055 24/4/2012 Machens et al, Genetic World J testing Surg 2007;31:957-968 in MTC 39
Codon specific Age related MTC progression in familial syndromes Codon 609,611,618,620,630,63 4(n=165) 768,791,804,891(n=27) CCHP Mean Age Node ve MTC Node +ve MTC P value 8.3 10.2 17.1 <0.001 11.2 16.6 - = 0.005 *918(n=17) - 3.0 16.0 0.069 634(n=129) 6.9 10.1 16.7 <0.001 *Codon 918,611, 791 no stastical difference 609,630,891- No or scarce data hence needs further verification Study 1- n=207 age<20,t<1.0cm Study 2 n=167 Machens etal. N Engl J Med 2003; 349:1517-1525 *Machens etal. Surgery 2003;134:425-431 24/4/2012 Genetic testing in MTC 40
RET Mutations RET mutation correlates with the phenotypic expression of MEN 2 Pheo & HPT : M.C with Codon 634 mutations HPT : does not occur in codon 918 mutation M.C mutation : mis-sense mutation at codon 634 in change from cysteine to arginine RET mutations stratified into 3 groups on the aggressiveness of MTC Codon 609, 768, 790, 791, 804 & 891 Codon 611, 618, 620 & 634 Codon 883 & 918 Yip L et al, Arch Surg 2003; 138: 409-416 41 Genetic testing in MTC 24/4/2012
Age of Manifestation Codon Age of MTC Age of Lymph node Mets Highest Risk 883,918,922 Age of distant metastasis < 1yr 2.5 yrs 5 yrs High Risk 611,618,620,634 <1yr (634) <5 yrs (rest) 1 st and 2 nd Decade 3 rd Decade Low Risk 609,768,790,804,891 2 nd to 3 rd decade 2nd to 4 th decade >4 th Decade Machens etal. World J Surg 2007;31:957-968 42 Genetic testing in MTC 24/4/2012
Prophylactic Thyroidectomy Risk Highest risk (Level 3) High risk (Level 2) Least aggressive (Level 1) Solution TT+ CCLND within 6 months of age preferably within 1 month TT +/- CCLND before 5 yrs of age Between 5-10yrs TT Brandi etal. J Clin Endocrinol Metab, 2001;86(12)5658-5671 43 Genetic testing in MTC 24/4/2012
ATA Risk Level & Prophylactic Thyroidectomy testing & Therapy ATA risk level D (918,883) C (634) Age of RET testing ASAP & within 1 st yr of life Age of required first USG ASAP & within 1 st yr of life Age of required first serum Ct 6 months, if surgery not already done Age of Prophylactic Thyx ASAP & within the 1 st yr of life <3-5 yrs >3-5 yrs >3-5 yrs Before age 5 yrs B (620,630, 609, 611,618) A (804,790, 791 ) <3-5 yrs >3-5 yrs >3-5 yrs Consider Surgery before age 5. May delay surgery beyond 5 yrs of age if stringent criteria are met <3-5 Yrs >3-5 yrs >3-5 yrs May delay Surgery beyond age 5 yrs if stringent criteria are met Stringent Criteria : normal annual basal+/- stimulated serum Ct, normal neck USG, less aggressive MTC family history, & family preference ASAP = as soon as possible Kloos TR et al, Management Guidelines of the ATA, Thyroid;19:6,2009 : 565-612 44 Genetic testing in MTC 24/4/2012
Current Recommendation for screening All Cases of apparently sporadic MTC should be tested for germline mutation Mutations involving RET exons 10,11,13,14,15 and 16 should be tested routinely If these are negative then test for additional 15 Exons Exons 13,14 & 15 should be screened carefully as they have low incidence of PCC hence prone for missing the syndromic association Screening in kindred can be for the specific codon mutation detected in Proband Tests in tumour tissue for somatic RET mutation in sporadic MTC or in sporadic PCC are generally not recommended for clinical use Brandi etal. J Clin Endocrinol Metab, 2001;86(12)5658-5671 45 Genetic testing in MTC 24/4/2012
Current Recommendation for screening Basal or stimulated calcitonin immuno-assays is used to monitor tumour status for recurrence / persistence / or metastasis. RET testing should be considered for <50 yrs onset and bilateral PCC Periodic screening for tumours in MEN 2 carrier is based upon the MEN 2 variant, as characterized by the codon mutation and by manifestations in the rest of the family members Brandi etal. J Clin Endocrinol Metab, 2001;86(12)5658-5671 46 Genetic testing in MTC 24/4/2012
47 Genetic testing in MTC 24/4/2012
Clinical implications of RET mutations 48 Machens A et al, NEJM 2003,349: 1517-25 Genetic testing in MTC 24/4/2012
Genetic testing in diagnosis and management of MEN Patient with MTC (Index case) Germline RET mutation analysis RET +ve patient (hereditary disease) RET mutation analysis in all first degree relatives Ret ve patient / Not done Pentagastrin test in 3 first degree relatives RET negative RET positive Negative pentagastrin test-insignificant risk No further evaluation Surgery Positive Surgery For those refusing surgery / subjects with exon 13,14,15 mutations Pentagastrin test Negative Annual pentagastrin test 49 Genetic testing in MTC 24/4/2012
Summary RET mutation testing should be done for all sporadic cases of Medullary thyroid carcinoma Genetic Screening is a reliable test for detection of at risk individuals and will help to avoid further screening of not at risk kindreds(50%) Timing of Surgery may be different depending on the risk categorization of the codon mutations Timing for Screening of associated components of MEN 2 differs with different codon mutations due to the variation in association. Genetic Counseling is an essential part of managing these patients 50 Genetic testing in MTC 24/4/2012