Universitätsklinikum Carl Gustav Carus. Graeme Eisenhofer

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1 Universitätsklinikum Carl Gustav Carus DIE DRESDNER. Biochemistry of Phaeochromocytoma Graeme Eisenhofer Institut für Klinische Chemie und Laboratoriumsmedizin and Medizinische Klinik III, Universitätsklinikum Carl Gustav Carus Dresden, Germany AACB Sydney

2 Shifting Emphasis of Laboratory Tests for Biochemical Diagnosis Colormetric Assays 1950 Improved understanding catecholamine metabolism 1990 HPLC Assays 2000 Shift from catecholamines to metanephrines for biochemical diagnosis 2015 LC-MS/MS (routine) Urinary Catecholamines Plasma free metanephrines Urinary total metanephrines Urinary fractionated metanephrines Urinary fractionated metanephrines Urinary catecholamines Urinary VMA Plasma catecholamines Plasma CgA Spectrophotometric assays Radioenzymatic assays, HPLC Plasma catecholamines Plasma CgA Urinary VMA LC-MS/MS RIA & EIA HPLC

3 IJ Kopin LL Iversen J Axelrod US von Euler A Carlsson Axelrod J. The fate of noradrenaline in the sympathetic neurone. Harvey Lect. 1973;67: U Trendelenburg A Sjoerdsma M Henseling SE Gitlow SZ Langer JW Maas H. Bönisch JR Crout KH Graefe EH La Brosse M Sandler CR Creveling RJ Wurtman S Udenfriend GM Tyce RD Hoeldtke

4 Model quantitatively illustrating release, uptake, metabolism and turnover of noradrenaline in the human heart at rest Extraneuronal uptake and metabolism? Eisenhofer G, Esler MD, Meredith IT, Dart A, Cannon RO 3rd, Quyyumi AA, Lambert G, Chin J, Jennings GL, Goldstein DS. Sympathetic nervous function in human heart as assessed by cardiac spillovers of dihydroxyphenylglycol and norepinephrine. Circulation :

5 1992 Karel Pacak Jacques Lenders

6 Sympathetic nerve varicosity Bloodstream MAO NE Adrenalmedullary cell DHPG NE MAO 93% NE NE COMT NMN 7% 77% NMN MAO E COMT Extraneuronal cell MN E 9% MN MHPG NE 23 % 95% 91% NE COMT NMN PNMT E MN E COMT

7 A recurring observation: normal plasma catecholamines with elevated metanephrines Plasma concentration (nmol/l) MEN 2 patient with pheochromocytoma Norepinephrine Normetanephrine Epinephrine Metanephrine 4 2 URL NE URL E 1 URLNMN 0 URLMN 0 GLUC 1 2 Day 3 GLUC 1 2 Day 3

8 1964 We concluded that in some pheochromocytomas much of the catecholamine synthesized is degraded directly in the tumor before it ever reaches the circulation Noted differences in tumors that produced only norepinephrine from those that produced both epinephrine and norepinephrine Metabolism Secretion

9 Morris Brown Jacques Lenders Harry Keiser Dick Crout Jacques Willemsen Bill Manger Gyorgy Czacko Bob Peaston Ravinder Singh Graeme Eisenhofer Patti Sullivan

10 . Endocr Pathol Fall;14(3):

11 Performance of Biochemical Tests for Diagnosis of Hereditary and Sporadic Pheochromocytoma Sensitivity Hereditary Sporadic (76) (138) Plasma Tests (n) Free metanephrines Catecholamines Urine Tests (n) Fract. metanephrines Total metanephrines Catecholamines VMA 97% 69% (68) 96% 60% 79% 46% Specificity Hereditary Sporadic (339) (305) 99% 92% 96% 89% 82% 72% (107) (324) (211) 97% 88% 91% 77% 82% 97% 96% 99% 45% 89% 75% 86% From: Biochemical Diagnosis of Pheochromocytoma: Which Test Is Best? JAMA. 2002;287:

12 Summary of 20 studies of plasma free metanephrines for diagnosis of pheochromocytoma Study - Authors - yr 1. Raber et al, Lenders et al, Sawka et al., Unger et al., Giovanella et al., Vaclavik et al, Gao et al., Hickman et al., Procopiou et al., Grouzmann et al., Peaston et al., Mullins et al, Sarathi et al, Christensen et al., Unger et al., Pussard et al, Därr et al, Därr et al, Tanaka et al., Weismann et al, Kim et al., 2014 TOTAL Analytical method LC-ECD LC-ECD LC-ECD RIA LC-ECD LC-ECD EIA LC-ECD EIA LC-ECD LC-MS/MS EIA EIA EIA EIA RIA LC-MS/MS LC-MS/MS EIA LC-MS/MS LC-MS/MS Sampling position Supine Supine Seated Seated Not stated Supine Supine Not stated Not stated Supine Seated Seated Seated Seated Seated Supine Seated Supine Seated Supine Seated Total Patients No with tumors Sensitivity Specificity 100% 99% 97% 96% 95% 100% 97% 100% 91% 96% 100% 100% 94% 91% 90% 100% 97% 100% 96% 100% 96% 100% 89% 85% 79% 94% 97% 86% 98% 100% 89% 96% 88% 94% 99% 90% 96% 71% 95% 97% 99% 76% 98% 90%

13 1.1 We recommend that initial biochemical testing for PPGLs should include measurements of plasma free metanephrines or urinary fractionated metanephrines. (1++++) 1.2 We suggest using liquid chromatography with mass spectrometric or electrochemical detection methods rather than other laboratory methods to establish a biochemical diagnosis of PPGL. (2++ ) J Clin Endocrinol Metab, June 2014, 99(6): For measurements of plasma metanephrines, we suggest drawing blood with the patient in the supine position and use of reference intervals established in the same position. (2++ ) 1.4 We recommend that all patients with positive test results should receive appropriate follow-up according to the extent of increased values and clinical presentation. (1++ )

14 Endocrine Society recommendation for initial biochemical testing 1.1 Initial biochemical testing for PPGLs should include measurements of plasma free metanephrines or urinary fractionated metanephrines..one of the most important considerations in choice of initial test is a high level of reliability that the test will provide a positive result in that rare patient with the tumour. This conversely also provides confidence that a negative result reliably excludes the tumour, thus avoiding the need for multiple or repeat biochemical testing or even costly and unnecessary imaging studies to rule out the tumour.

15 Supine versus seated blood sampling for plasma metanephrines Avoidance of posture-independent forms of stress (e.g., emergency or ICU conditions and never during a hypertensive crisis) Diet (e.g., overnight fast methoxytyramine; curry for urine deconjugated metanephrines by HPLC) Medication-causes of false-positive results (e.g., tricyclic antidepressants) Appropriate reference intervals (e.g., age-adjusted reference intervals)

16 Seated versus supine sampling Historically tests of plasma metanephrines for diagnosis were set up and validated with supine sampling, this recognizing the profound impact of upright posture on sympatho-adrenal function. Fasting because of dietary chromatographic interferences. These precautions are not commonly followed!

17 Summary of 20 studies of plasma free metanephrines for diagnosis of pheochromocytoma Study - Authors - yr 1. Raber et al, Lenders et al, Sawka et al., Unger et al., Giovanella et al., Vaclavik et al, Gao et al., Hickman et al., Procopiou et al., Grouzmann et al., Peaston et al., Mullins et al, Sarathi et al, Christensen et al., Unger et al., Pussard et al, Därr et al, Därr et al, Tanaka et al., Weismann et al, Kim et al., 2014 TOTAL TOTAL TOTAL Analytical method LC-ECD LC-ECD LC-ECD RIA LC-ECD LC-ECD EIA LC-ECD EIA LC-ECD LC-MS/MS EIA EIA EIA EIA RIA LC-MS/MS LC-MS/MS EIA LC-MS/MS LC-MS/MS Sampling position Supine Supine Seated Seated Not stated Supine Supine Not stated Not stated Supine Seated Seated Seated Seated Seated Supine Seated Supine Seated Supine Seated Supine Seated Total Patients No with tumors Sensitivity Specificity 100% 99% 97% 96% 95% 100% 97% 100% 91% 96% 100% 100% 94% 91% 90% 100% 97% 100% 96% 100% 96% 100% 89% 85% 79% 94% 97% 86% 98% 100% 89% 96% 88% 94% 99% 90% 96% 71% 95% 97% 99% 76% % 95% % 96% 90% 85%

18 Compliant versus non-compliant sampling: Patients without pheochromocytoma 64% 49% Compliant Non-compliant P< % 14% Compliant Two compliant centers (n=438) Non-compliant Plasma methoxytyramine (nmol/l) P<0.001 Plasma metanephrine (nmol/l) Plasma normetanephrine (nmol/l) Upper cut-offs determined by 97.5 percentiles considerably higher for samples collected in seated position without fasting than in supine position with fasting P< % 51% Compliant Non-compliant Four non-compliant centers (n=195) Clin Endocrinol (Oxf) Apr;80(4):

19 Compliant versus non-compliant sampling: Patients with pheochromocytoma Among patients with phaeochromocytoma, plasma concentrations for samples collected in the seated position without fasting are not higher than in supine position with fasting Compliant Non-compliant Two compliant centers (n=62) NS Plasma methoxytyramine (nmol/l) P<0.05 Plasma metanephrine (nmol/l) Plasma normetanephrine (nmol/l) NS Compliant Non-compliant Compliant Non-compliant Four non-compliant centers (n=67) Clin Endocrinol (Oxf) Apr;80(4):

20 Impact of seated, non-fasting sampling on plasma metanephrine diagnostics Loss of upper cut-offs determined Loss of of diagnostic diagnosticsensitivity specificitywith withuse seated non-fasting samplings Diagnostic sensitivity and specificity for compliant and non-compliant from seated non-fasting samplings using upper cut-offs determined byupper supine fasted samplings conditions of blood sampling according to cut-offs for supine and fasting (9-fold increase in results) (6-fold increase in false false negative positive results) conditions versus seated and non-fasting conditions Sensitivity Specificity (All data) Supine / fasting Seated / non-fasting Cut-offs (n=130) (n=438) (n=195) Supine and fasting 98.5% 95.0% 70.3% (128/130) (416/438) (137/195) 85.4% 99.8% 94.9% (111/130) (437/438) (185/195) Seated and non-fasting Clin Endocrinol (Oxf) Apr;80(4):

21 ROC curve analysis of diagnostic test performance Plasma metanephrines supine vs seated vs urine fractionated metanephrines 100% Sensitivity 96% Specificity Sensitivity 92% Sensitivity 95% Specificity 87% Sensitivity 92% Specificity Plasma - Compliant AUC = Urine Frac. NMN,MN,MTY AUC = Plasma - Non-compliant AUC = % 80% 60% 40% 20% Specificity 0% Clin Endocrinol 2014; 80:

22 24 hr urine deconjugated metanephrines 24 hr urines Acid hydrolysis deconjugation step Significant potential for underestimated outputs and missed diagnoses Difficult to control for factors that increase outputs (diet, sympathetic activation etc) and cause false-positives

23 Urine free metanephrines Boyle JG et al J Clin Endocrinol Metab Dec;92(12): Whiting MJ. Ann Clin Biochem Mar;46(Pt 2): Peitzsch M, et al. Clin Chim Acta Mar 15;418:50-8. Free DA MN NMN NA A MTY Deconjugated

24 Overnight urine collections? Free urinary metanephrines and catecholamines corrected for creatinine Noradrenaline Normetanephrine Adrenaline Metanephrine Simpler than 24 hour urine collections, but do overnight or first morning urines provide similar or even improved diagnostic accuracy over 24 hour collections?

25 Reference intervals for overnight (first morning) urine free metanephrines Urine outputs expressed as µmol/mmol creatinine Urine free normetanephrine Urine free metanephrine NS P F F M M Females Males Females Males 254 healthy, normotensive and hypertensive volunteers (155 females, years; 99 males, years)

26 Diagnostic test performance for overnight (first morning) urine free metanephrines Sensitivity 24 hr urine deconjugated metanephrines [µg/24h] 24 hr - urine free metanephrines [µg/24h] Overnight - urine free metanephrines [µmol/mmol creatinine] Specificity 36/38 336/ % 81.4% 37/38 372/ % 89.2% 37/38 396/ % 95.0% 455 patients tested for phaeochromocytoma including 38 with tumours

27 Reference intervals: plasma free metanephrines Should only be determined from reference populations sampled in the supine position At the NIH 97.5 percentiles for normetanephrine determined at 0.61 nmol/l (112 pg/ml) At the Mayo medical laboratories 0.90 nmol/l (165 pg/ml) - but for seated samples (~50% higher than for NIH) Age considerations Harmonization via QA programs

28 Age-adjusted reference intervals for plasma normetanephrine (LC-ECD data) Historical reference data (n=1226) Laboratory data (n=3888) 96.0% Sensitivity Specificity 93.9% 93.9% 93.6% 93.7% 93.6% 91.2% 88.3% No pheochromocytoma (n = 558) 3330) Pheochromocytoma

29 Age-adjusted reference intervals for plasma normetanephrine (LCMS/MS data) Reference population (n=299) nmol/l Upper cut-off defined by y = x In nmol/l y = 2.075x10-6 x

30 pg/ml Reference intervals for plasma metanephrine (LC-MS/MS data) 97.5 percentile 62 pg/ml (0.31 nmol/l) 99.5 percentile 84 pg/ml (0.43 nmol/l)

31 pg/ml Reference intervals for plasma methoxytyramine (LC-MS/MS data) 97.5 percentile 11 pg/ml (0.067 nmol/l) 99.5 percentile 16 pg/ml (0.098 nmol/l)

32 Harmonization: RCPA QA Program Immunoassays % Lower!

33 Standardization among laboratories: RCPA QA Program - Immunoassays It appears that free NMN were systematically lower with the immuno-assay independent of the matrix

34 EIA vs LC-MS/MS for Plasma Normetanephrine Difference LC-MS/MS EIA [%] EIA Normetanephrine [pg/ml] 341 Patients tested for phaeochromocytoma, including 54 with tumours LC-MS/MS Normetanephrine [pg/ml] 60% lower by EIA Mean of LC-MS/MS and EIA Normetanephrine [pg/ml] Eur J Endocrinol Mar;172(3):

35 Diagnostic test performance: LC-MS/MS vs EIA according to reference intervals LC-MS/MS Sensitivity Upper cut-offs NMN 0.54 to 1.08 nmol/l* MN 0.42 nmol/l 100% MTY 0.10 nmol/l (54/54) EIA Sensitivity Package insert upper cut-offs NMN 0.98 nmol/l 74.1% (40/54) MN 0.46 nmol/l Specificity 98.6% (283/287) Specificity 98.9% (284/287) Bias corrected upper cut-offs NMN 0.22 to 0.44 nmol/l* 96.2% MN 0.27 nmol/l (273/287) (52/54) 95.1% * Age adjusted upper cut-offs for NMN

36 mk22 Plasma free metanephrines: Prediction of tumour location and size Metanephrine: normetanephrine+metanephrine ratio > 10% indicates adrenal location Clinical Chemistry (4):

37 Slide 36 mk22 Insert your project number here (Aria 32, bold, white). Delete comment. mk-user, 10/02/2011

38 Genetics MDH2 FH Genetics timeline for chromaffin cell tumors HIF2 PGL MEN 2 NF1 VHL VHL RET MAX >30% tumors hereditary SDHA rule-of-ten defunct TMEM127 SDHD SDHAF2 SDHC SDHB NF Fränkel NF1 (neurofibromatosis type1) SDHD (PGL1) RET (MEN 2) SDHC (PGL3) VHL (von Hippel Lindau) SDHB (PGL4) SDHAF2 (PGL2) SDHA TMEM127 MAX HIF2 FH MDH2 19 identified tumor susceptibility genes >35% germline KIF1B PHD1&2 HRAS IDH1 ATRX 60% germline and somatic

39 PHD1&2 Cluster 1 VHL FH MDH2 SDHA SDHB SDHC SDHD SDHAF2 HIF2 Cluster 2 MAX TMEM127 NF1 RET HRAS Courtesy Karel Pacak J Natl Cancer Inst Sep 4;105(17):

40 RET 2 NF1 cluster EPI/NE EPI/NE <5% VHL 2 TMEM127 cluster 1 SDHB NE EPI/NE 8% cluster 1 SDHD cluster 2 cluster 10% 1 SDHA cluster /C/AF2 NE/DA 65% cluster NE>EPI <5% NE/DA 8% 2 MAX cluster NE/DA 25%? 1

41 mk19 Genotype biochemical phenotypes From: Clinical Chemistry 2011 Mar;57(3):411-20

42 mk20 Genotype biochemical phenotypes: 3D-plot MEN 2 & NF1 SDH VHL From: Clinical Chemistry 2011 Mar;57(3):411-20

43 Distinct catecholamine secretory profiles in cluster 1 versus cluster 2 tumours Tumor Tissue Catecholamine Contents (nmol/gm) Adrenaline Noradrenaline Dopamine CLUSTER VHL MEN2 SDHB SDHD 1 NF1 2 NA SPOR 1 A SPOR 2

44 Cluster 1 versus cluster 2 tumours differ in maturity of the regulatory secretory pathway Constitutive Secretory Pathway Regulated Secretory Pathway Spontaneous Ca2+-independent Rapid Continuous Not responsive to secretogogues Secretory vesicles Cluster 1 Noradrenergic Immature Syntaxin Nucleus EPI Norepinephrine vesicle Ca2+ Synaptotagmin DOPA NE DA SNAP25 DA Pathways more developed DBH Catecholamine Biosynthetic Pathway VAMP Ca2+ TH AADC NE Rabphilin3A Rab TYR PNMT Cluster 2 Adrenergic Mature Golgi Apparatus Contains chromogranins, NPY, Epinephrine and peptide processing vesicle enzymes EPI Ca2+- dependent Storage pool of vesicles held in cytoskeletal frame Signal Ca2+ Large dense core vesicles Vesicle sorting Peptide and protein packaging & pathway components more highly expressed in Cluster 2 SCAMP Ca2+ Other sensors CADPS Annexin A7 Ca2+ transducers CALM 1 & 2 Catecholamine Secretory Pathway

45 Cluster 1 versus cluster 2 tumours Chromaffin tumour progenitor cells Cluster 2 Cluster 1 HIF2α HIF2α HIF1α Nan Qin HIF1α Int J Cancer Nov; 135: HIF2α HIF1α HIF1α PHD Succinate SDHx Oxaloacetate Cluster 1 Pseudohypoxic Hypoxia-angiogenic pathways MDH2 Malate VHL HIF2α HIF1α Fumarate FH HIF2α HIF1α MYC MAX Differentiation arrested Immature phenotype Cluster 1 tumors TM mtor EM 12 7 Akt RET Ras HRas NF1 Differentiation permitted Mature phenotype Cluster 2 tumors Cluster 2 Kinase signalling RAS & mtor pathways

46 Succinate Dehydrogenase Proteosomal degradation -Ketoglutarate Isocitrate Succinate Pyruvate Citrate Acetyl-CoA PHD Succinyl-CoA Succinate -Ketoglutarate HIF2 OH OH X Succinate dehydrogenase Fumarate Oxaloacetate Malate HIF2 HIF1 HIF2 HRE regulated MYC MAX gene expression TET HDM DNA Myc regulated gene expression Histone demethylation demethylation Nucleus

47 SDHB-mutated tumors: high predisposition to malignancy No SDHB SDHB No SDHB SDHB No SDHB SDHB From: Eur J Cancer Jul;48(11): Plasma methoxytyramine: a novel biomarker of metastatic pheochromocytoma and paraganglioma in relation to established risk factors of tumour size, location and SDHB mutation status.

48 Most likely location Adrenal or adrenal recurrence Adrenal or extra-adrenal Adrenergic MN NMN Extra-adrenal Noradrenergic MTY MN RET / NF1/ TMEM127 NMN MTY VHL / SDHx Dopaminergic MN NMN MTY SDHx Most likely mutation decreasing phenotypic maturity increasing malignant risk From: Clin Chem Dec;60(12):

49 Key points Utility beyond diagnosis Urine into Insights metanephrines chromaffin tumour biology Deconjugated (e.g., cluster 1 &vs2 free tumours) Plasma metanephrines Tool to guide cost-effective genotyping of underlying Supine versus disease-causing mutations Seated Sampling Biochemical diagnosis of chromaffin cell tumours Understanding of catecholamine metabolism metanephrines, metanephrines, metanephrines Use of appropriate reference intervals Urine metanephrines Assessment of disease burden Possible alternatives over-night or Surrogate biomarkers first morning of therapeutic response PlasmaFor methoxytyramine: plasma biomarker of malignancy metanephrines Age-adjustments Disease stratification

50 Key points Interpretation of biochemical test results Utility beyond diagnosis Tumour localization (e.g., cluster 1 & 2 tumours) extra- vs intra-adrenal & choice of functional imaging modality Cluster 1 VHL (NMN) SDH (MTY & NMN) Cluster 2 MEN2/NF1/ TMEM127 (MN & NMN) Chromaffin cell tumours: distinct biochemical profiles dependent on underlying mutations - associated with biological behaviour Patient management Age to start screening in hereditary cases Follow-up - risk of malignancy - MTY, size, location SDHB Selection of therapy for metastatic disease Personalised medicine