clinical study cushing s disease: the relevance of a

Clinical StudyCushingrsquos Disease The Relevance of a CombinedDexamethasone Desmopressin Test as a Component ofPostoperative Hormonal Evaluation

PrzemysBaw Witek1 Grzegorz ZieliNski2 and Katarzyna Szamotulska3

1Department of Endocrinology and Isotope Therapy Military Institute of Medicine Ul Szaserow 128 04-141 Warsaw Poland2Department of Neurosurgery Military Institute of Medicine Ul Szaserow 128 04-141 Warsaw Poland3Department of Epidemiology Institute of Mother and Child Ul Kasprzaka 17a 01-211 Warsaw Poland

Correspondence should be addressed to Przemysław Witek pwitekwimmilpl

Received 6 April 2015 Revised 2 June 2015 Accepted 9 June 2015

Academic Editor Elena Valassi

Copyright copy 2015 Przemysław Witek et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Background The risk of Cushingrsquos disease (CD) recurring may persist for years even after initially successful surgery ObjectiveTo prospectively assess the relevance of a combined dexamethasone desmopressin test (CDDT) as a component of postoperativehormonal evaluation including the dynamics of ACTH and cortisol concentrationsMaterial andMethodsWe included 28 patientsafter TSS for CD Eighteen months after surgery the standard hormonal evaluation was performed followed by a CDDT ResultsFifteen patients (536) were in remission whereas in 13 subjects (464) hypercortisolemia was confirmed Positive results ofCDDT were observed in 12 noncured patients (923) and in one subject in remission (67) Negative results were obtained in12 patients with remission (80) and in one noncured patient (77) With 2 patients in CD remission (133) the test resultswere inconclusive We confirmed a high compatibility between CDDT and standard hormonal assessment results (120581 = 0846119875 lt 0001) Significant differences in ACTH and cortisol levels at each CDDT time point between the two studied subgroups wereshown Conclusions A negative CDDT result can be regarded as one of the factors indicative of CD remission during follow-upAdditionally CDDT can help distinguish persistent hypercortisolemia from naturally recurring adrenal function after TSS

1 Introduction

The diagnosis and treatment of Cushingrsquos disease (CD) areamong the greatest challenges in endocrinologyThediagnos-tic criteria of CD and the treatment principles in typical caseshave been established according to a consensus of experts[1 2] The treatment of choice is transsphenoidal selectiveadenomectomy performed by an experienced neurosurgeonHowever postoperative management protocols differ acrosscentres and countries [3ndash5] Even after initially effectivetranssphenoidal removal of a corticotroph adenoma the 20ndash30 risk of recurrence even many years later remains amajor concern [6]

Some centres use a stimulation test with the syntheticvasopressin analog desmopressin (1-deamino-8D-argininevasopressin DDAVP) as a means of differentiating CD

from both pseudo-Cushing states and the ectopic ACTH-dependent Cushingrsquos syndrome Some authors suggested theuse of this test during postoperative follow-up with a persis-tent response to desmopressin (positive test result) indicativeof persistent Cushingrsquos disease or a higher risk of recurrence[7ndash10] Castinetti et al suggested the use of a combined 1mgdexamethasone suppression and desmopressin stimulationtest in evaluating the risk of hypercortisolemia recurrence[9]The addition of dexamethasonewas to increase specificityof the test by suppressing the normal corticotroph cells ofthe pituitary while stimulating only residual tumor cellsHowever a desmopressin test is not typically used in routinepostoperative diagnostics In accordance with the EndocrineSociety recommendations it should only be performed aspart of research studies [1 2]

Hindawi Publishing CorporationInternational Journal of EndocrinologyVolume 2015 Article ID 357165 8 pageshttpdxdoiorg1011552015357165

2 International Journal of Endocrinology

The aim of this study was to conduct a prospectiveevaluation of the role and usefulness of the combined dex-amethasone desmopressin test in postoperative assessmentsof transsphenoidal surgery effectiveness in CD patients in asingle pituitary center

2 Material and Methods

21 Patient Population The study population comprised 28consecutive patients with Cushingrsquos disease (22 women and6 men F M ratio 37 1) hospitalized and operated in theMilitary Institute of Medicine in Warsaw between 2010 and2012 The mean age was 361 plusmn 135 years (168ndash576) Aftertheir confirmed diagnosis of Cushingrsquos disease the patientswere operated on by the same neurosurgeon using the samesurgical protocol

All patients were informed about the aims and methodsof the study and they signed the informed consentThe studyprotocol was approved by the local ethics committee

22 Preoperative Endocrine Evaluation All patients under-went a standard clinical evaluation The diagnosis of ACTH-dependent Cushingrsquos syndrome (CS)wasmade based on clin-ical signs and standard hormonal criteria increased urinaryfree cortisol (UFC) increased morning serum cortisol levelat 800 loss of cortisol circadian rhythm (the serum cortisollevel above 75 120583gdL in the late-night hours (2200ndash0000))and increased or detectable level of plasma ACTH at 800and the failure of serum cortisol to suppress to less thanor equal to 18 120583gdL during the low dose dexamethasonesuppression test (LDDST 05mg qid for 48 hours) Thepituitary etiology of CS was confirmed based on serumcortisol or UFC suppression greater than 50 with thehigh dose dexamethasone suppression test (HDDST 2mgqid for 48 h) and positive pituitary MRI In the case ofinconclusive hormonal assessments and pituitary imagingthe diagnosis of Cushingrsquos diseasewas confirmed by a positiveresult of a stimulation test with intravenous CRH injection(100 120583g) andor results of inferior petrosal sinus sampling(IPSS)

23 Preoperative MRI Prior to neurosurgical treatmentall patients underwent high-resolution magnetic resonanceimaging of the pituitary-hypothalamic region (SIEMENSSymphony 2004 15 Tesla) performed before and after intra-venous injection of gadolinium (Gd-DTPA) The presencesize and position of any focal lesions were recorded Ahypodense lesion visualized after contrast administrationwasconsidered to be indicative of pituitary adenoma Microade-noma was defined as a pituitary tumor with a diameter of lessthan 1 cm whereas macroadenoma was defined as a pituitarytumor with at least one diameter of more than 1 cm

24 Surgical Procedure A microsurgical transseptal trans-sphenoidal approach was used for resection of ACTH-secreting pituitary adenomas The pituitary gland was care-fully examined regardless of MRI findings Selective ade-nomectomy was performed in all cases of MRI-visualized

pituitary adenomas Where MRI findings were inconclusiveor no tumor was evident a series of vertical and horizontalincisions of the pituitary gland were performed with all tissuedeemed abnormal removed and submitted for pathologicalexamination

25 Histopathological and Immunohistochemical AssessmentSurgical specimens were collected for histopathological anal-ysis (standard hematoxylin and eosin staining) and immuno-histochemical staining for pituitary hormones (kits withpolyclonal antibodies Dako Denmark) The result of ahistopathological assessment was considered to be positiveif the presence of corticotroph adenoma as well as positiveACTH-staining was confirmed in histological and immuno-histochemical examinations The result was treated as nega-tive if there was no histopathological evidence of corticotrophadenoma and ACTH-staining was negative

26 Postoperative Hormonal Evaluation and Criteria of CureBlood samples for serum cortisol measurements were col-lected from all patients at 600 on the first postoperativeday Glucocorticoids were not administered in the periop-erative or in the early postoperative period Hydrocortisonereplacement therapy was started after biochemical confir-mation of hypocortisolemia or the development of clinicalmanifestations of adrenal insufficiency The standard dose ofhydrocortisone (20mg in the morning and 10mg at 1500)was introduced when necessary and continued until the nexthormonal evaluation

Following the surgical procedure all patients weresubjected to further postoperative evaluations Subsequentreassessments were performed at 6 weeks and at 3 6 1218 and 24 months after surgery and once yearly thereafterPatients on hydrocortisone replacement therapy had theircortisol measurements taken 48 hours after the last admin-istered dose We used the following criteria for sustainedremission biochemical evidence of eucortisolemia (morningserum cortisol in the 5ndash25 120583gdL range) preserved circadianrhythm of serum cortisol (the late-night serum cortisol levelle75 120583gdL) and the ability of serum cortisol to suppressto less than or equal to 18 120583gdL after a 1mg overnightdexamethasone suppression test (ODST)

27 Combined Dexamethasone Desmopressin Testing Thecombined dexamethasone desmopressin test (CDDT) wasconducted in two stages After the informed consent had beensigned each patient received 1mg of oral dexamethasoneat midnight In patients who had received hydrocortisonereplacement therapy it was discontinued 48 hours prior tothe test A 10 120583g of desmopressinmdashMinirin (Ferring GmbHGermany)mdashwas administered as a slow intravenous bolus8 hours after dexamethasone administration Blood samplesfor testing ACTH and cortisol levels were collected beforeand 15 30 60 90 and 120 minutes after desmopressinadministration

Apositive test result which indicates persistent impropercorticotroph secretory response to desmopressin requiresplasma ACTH increase by at least 30 and serum cortisol

International Journal of Endocrinology 3

by at least 20 compared to baseline [7] The baseline valuescorrespond to plasma ACTH and serum cortisol followingODST In addition due to low ACTH and cortisol levels inpatients after successful TSS we adopted an additional crite-rion necessary for interpreting the test result as positive Thiswas an absolute increase in plasma ACTH by ge10 pgmL andserum cortisol by ge2 120583gdL An increase only in ACTH levelswithout a parallel increase in cortisol levels was interpretedas inconclusive

28 Hormone Assay Chemiluminescent immunometricassays (IMMULITE 2000 Siemens UK) were used tomeasure serum cortisol Method sensitivity was 02120583gdL(55 nmolL) and the reference range 50ndash250120583gdL Thequantification threshold for serum cortisol levels usingthe analyser was the measured value of 10 120583gdL Thusfor the purpose of further calculations it was assumed thatin these cases the postoperative cortisol was 10 PlasmaACTH levels were measured using a specific two-stepradioimmunometric assay (IRMA coated tube techniqueBrahms Germany) Method sensitivity was 12 pgmL(reference range 10ndash60 pgmL)

29 Statistical Analysis The statistical analysis employedmethods of descriptive statistics (mean median standarddeviation and proportion) The hypotheses concerning therelationship between two categorical variables were verifiedusing the exact chi-square test (Fisherrsquos exact test) Thesignificance of the differences between average values ofcontinuous variables in two groups was analyzed bymeans ofthe Mann-Whitney 119880 test for small samples Verification ofhypotheses concerning comparisons of the analyzed param-eters in two time points was conducted using the Wilcoxontest for small samples

The dynamics of analyzed tests results was conductedby calculating areas under the curve (AUCs) using thetrapezoid rule The significance of differences between AUCswas checked using the Mann-Whitney 119880 test In orderto present average values and dispersion of AUCs meanand standard errors of logarithmically transformed valueswere used The transformation was performed to obtain thenormal distribution of AUCs

The level of significance was set at 119875 lt 005 Thecalculations were made using the commercially availablestatistical software package SPSS v180

3 Results

Thirty-six consecutive patients after transsphenoidal surgeryfor CD participating in the structured postoperative follow-up program were offered an opportunity to undergo aCDDT 18 months after TSS A total of 28 patients (778)consented including 15 (out of a total of 23) patients (652)who were in remission of hypercortisolemia at 18 monthsfollowing surgery and all of the 13 patients with persistenthypercortisolemia Out of the 8 patients considered to be inremission who did not undergo the test there were 3 pregnant

Table 1 Basic pre- and postoperative characteristics of the studygroup

Age1 (years) 361 plusmn 135Males 119899 () 6 (214)BMI1 (kgm2) 296 plusmn 58Preoperative plasma ACTH2 (pgmL) 860 (620ndash1370)Preoperative serum cortisol1 (120583gdL) 267 plusmn 69Serum cortisol after HDDST2 (120583gdL) 39 (22ndash100)Initial (1st) TSS 119899 () 21 (750)Tumor size 119899 ()Microadenoma 23 (821)Macroadenoma 5 (179)

Immunohistochemical confirmation ofcorticotroph tumor 119899 () 21 (75)

Nadir serum cortisol2on 1st or 2nd postoperative day (120583gdL) 23 (12ndash82)

1Mean plusmn SD 2median (interquartile range)

or breastfeeding females and 5 patients who did not providetheir informed consent

31 Pre- and Perioperative Characteristics Detailed demo-graphic data preoperative characteristics of the study grouppreoperative and early postoperative hormone level resultsand immunohistochemical findings are presented in Table 1

32 Results of Postoperative Hormone Assessments Eighteenmonths after TSS themean serumcortisol levels at 800 in theentire study group were 148 plusmn 77 120583gdL (median 14 range53ndash324 120583gdL) Normal circadian rhythms of serum cortisolwere found in 17 patients (61) Mean serum cortisol levelsin an overnight dexamethasone suppression test (ODST) inthe whole group were 408 plusmn 423 120583gdL (median 17120583gdLrange 10ndash178 120583gdL) According to the adopted criteria 15patients (536) with serum cortisol levels at 800 withinthe referral range normal circadian rhythm and post-ODSTcortisol levels below 18 120583gdL were found to be in remissionof hypercortisolemiaThe remaining 13 patients (464) wereconsidered to have persistent hypercortisolemia In thesetwo patient subgroups mean serum cortisol levels at 800were 101 plusmn 35 120583gdL (median 102 range 53ndash164120583gdL)and 203 plusmn 76 120583gdL (median 168 range 116ndash331 120583gdL)respectively 119875 lt 0001 Mean cortisol levels after ODSTwere117 plusmn 024 120583gdL (median 10 range 1ndash17 120583gdL) and 746 plusmn414 120583gdL (median 77 range 32ndash178 120583gdL) respectively119875 lt 0001 Mean nadir serum cortisol levels on the 1stor 2nd postoperative day (determined retrospectively) inthe remission and uncured subgroups were 15 120583gdL plusmn 05(median 16 range 1ndash25120583gdL) and 140plusmn114 (median 87range 41ndash370) respectively 119875 lt 0001

33 Results of CDDT At first CDDT results were analyzedqualitatively According to the predetermined criteria thetest results were positive in 13 out of 28 patients (464)negative in 13 patients (464) and inconclusive in 2 patients


Table 2 Comparison of ACTH (pgmL) and cortisol levels (120583gdL) in the remission and uncured subgroups following CDDT

Remission (119899 = 15) Persistent CD (119899 = 13)119875 value

Mean plusmn SD Median Range Mean plusmn SD Median RangeCortisol (120583gdL)

01015840 127 plusmn 057 10 10ndash29 772 plusmn 637 48 24ndash257 lt0001151015840 208 plusmn 332 10 10ndash140 1643 plusmn 780 161 31ndash296 lt0001301015840 215 plusmn 352 11 10ndash148 2061 plusmn 872 229 30ndash327 lt0001601015840 186 plusmn 249 10 10ndash107 2222 plusmn 996 247 29ndash378 lt0001901015840 168 plusmn 180 10 10ndash80 2086 plusmn 1016 223 25ndash352 lt00011201015840 155 plusmn 155 10 10ndash70 1869 plusmn 1025 184 27ndash367 lt0001

ACTH (pgmL)01015840 354 plusmn 373 30 10ndash150 3431 plusmn 2954 190 90ndash960 lt0001151015840 856 plusmn 986 50 20ndash360 18562 plusmn 29756 910 200ndash11350 lt0001301015840 673 plusmn 734 40 10ndash250 16246 plusmn 23292 950 220ndash9000 lt0001601015840 470 plusmn 528 30 06ndash190 10115 plusmn 12860 730 160ndash5020 lt0001901015840 377 plusmn 401 20 04ndash160 8500 plusmn 11538 500 110ndash4410 lt00011201015840 329 plusmn 320 20 10ndash130 7269 plusmn 10418 340 70ndash3950 lt0001

(72) A comparison of the CDDT results with typicallyperformed hormonal assessments at month 18 following TSSrevealed positive test results in 12 out of 13 uncured patients(923) and in 1 out of 15 patients (67) considered tobe in remission after 18 months Negative test results wereobserved in 12 out of 15 patients (80) in remission ofCushingrsquos disease and in 1 out of 13 uncured patients (77)CDDT results proved to be highly consistent with surgicalefficacy assessments based on the conventional hormonalcriteria presented above (120581 = 0846 119875 lt 0001) The testwas inconclusive in 2 out of 15 patients (133) who methormonal criteria for remission of hypercortisolemia In bothcases only plasma ACTH elevation was observed without aconcomitant increase in serum cortisol levels

Test results were analyzed separately in the remissionsubgroup and in the uncured CD subgroup Analysis findingsare presented in Table 2 There were significant differences interms of cortisol levels between the remission and uncuredsubgroups (119875 lt 0001) at all evaluated time points Moreoverthe two subgroups differed significantly in terms of plasmaACTH levels in respective consecutive test time points (119875 lt0001)

Subsequently the remission and uncured groups werecompared in terms of absolute (Δ) and relative (Δ) changesin cortisol levels at different test time points versus baselinevalues A similar comparisonwas then conducted for absolute(Δ) and relative (Δ) changes in ACTH levels The groupin remission of hypercortisolemia and the uncured groupdiffered significantly in terms of cortisol levels at every timepoint Similar findings were shown in terms of ACTH levelsat every time point except for the relative change in minute 15of the test Detailed findings have been presented in Tables 3and 4

Six patients (462) from the uncured subgroup (119899 =13) reached maximum ACTH levels at minute 15 whilethe remaining 7 patients (538) reached maximum ACTHlevels at minute 30 The maximum increase in cortisol levelsfollowing the CDDT was observed in 1 patient (77) at

minute 15 in 2 patients (152) at minute 30 in 7 patients(539) at minute 60 in 2 patients (153) at minute 90 andin the case of 1 patient (77) at minute 120

Twopatientswhomet the criteria forCD remission and inwhom test results were inconclusive achieved the maximumplasma ACTH levels at 15 minutes One of these patientsachieved an increase in plasma ACTH levels from 15 pgmLat the starting point to 25 pgmL at 15 minutes and theother patient from 3 pgmL to 16 pgmL respectively Serumcortisol levels in both patients were below 10 120583gdL and didnot increase throughout the test

We also compared the two subgroups in terms of areasunder the curve (AUCs) for the tested ACTH and cor-tisol levels The AUCcortisol values in the remission anduncured subgroups were as follows mean 21846plusmn284 120583gdLmedian 1254 and range 120ndash123045120583gdL and mean234081 plusmn 10566 120583gdL median 259125 and range 33533ndash406575 120583gdL respectively (119875 lt 0001) The AUCACTHvalues in the remission and uncured groups were as followsmean 60995 plusmn 64252 120583gdL median 4275 and range 1245ndash22275 pgmL and mean 1337192 plusmn 184679 pgmL median8190 and range 1875ndash71805 120583gdL respectively (119875 lt 0001)Following a logarithmic transformation the individual logAUC values for ACTH and cortisol were compared andpresented asmean and standard error of themean (SEM)Themean individual log-transformedAUCcortisol values in theCDremission and uncured subgroups were as follows 509plusmn062(SEM016median 483 range 479ndash712) and 759plusmn071 (SEM02 median 786 range 582ndash831) respectively (119875 lt 0001)Themean individual log-transformedAUCACTH values in theremission and uncured groups were as follows 601 plusmn 09(SEM 023 median 606 range 482ndash771) and 895 plusmn 104(SEM 029 median 901 range 754ndash118) respectively (119875 lt0001) (see Figures 1 and 2)

Our results suggest that Cushingrsquos disease remissiongroup and persistent CD group differ significantly in termsof combined dexamethasone suppression and desmopressinstimulation test results The relative and absolute changes in


Table 3 Absolute (Δ) changes in cortisol and ACTH levels at consecutive time points compared to baseline stratified by remission status



151015840 081 plusmn 286 00 minus01ndash111 871 plusmn 620 98 02ndash203 lt0001301015840 089 plusmn 306 01 minus02ndash119 1290 plusmn 710 138 03ndash223 lt0001601015840 060 plusmn 201 00 minus02ndash78 1450 plusmn 780 140 03ndash248 lt0001901015840 041 plusmn 132 00 minus02ndash51 1314 plusmn 855 137 minus01ndash287 lt00011201015840 028 plusmn 107 00 minus01ndash41 1097 plusmn 877 92 minus08ndash326 lt0001

ACTH (pgmL)151015840 502 plusmn 717 20 00ndash270 15131 plusmn 29289 550 10ndash10930 lt0001301015840 319 plusmn 442 10 minus05ndash160 12815 plusmn 22738 560 30ndash8580 lt0001601015840 116 plusmn 208 05 minus10ndash50 6685 plusmn 12070 330 10ndash4600 lt0001901015840 023 plusmn 141 00 minus20ndash40 5069 plusmn 10612 250 20ndash3990 lt00011201015840 minus025 plusmn 151 00 minus30ndash30 3839 plusmn 9601 90 minus20ndash3530 0002

Table 4 Relative (Δ) changes in cortisol and ACTH levels at consecutive time points versus baseline stratified by remission status




ACTH (pgmL)151015840 1434 plusmn 1299 1000 00ndash4333 5391 plusmn 8820 1778 53ndash26024 0121301015840 910 plusmn 1037 467 minus250ndash3000 4617 plusmn 6732 1579 158ndash20429 0024601015840 275 plusmn 612 267 minus500ndash1667 2318 plusmn 3170 917 53ndash10952 0004901015840 114 plusmn 549 00 minus636ndash1333 1561 plusmn 2567 531 105ndash9500 00041201015840 15 plusmn 426 00 minus500ndash1000 1190 plusmn 2417 105 minus222ndash8405 0011

10

9

8

7

6

5

4

3

509

759

RemissionPersistent CD

ln AUCcortisol

Figure 1 The mean individual log-transformed cortisol area underthe curve (AUC) obtained in the CDDT in CD remission anduncured patients (119875 lt 0001) with standard errors of the mean(SEM)

cortisol andACTH levels were evaluated in both subgroups atevery test time pointThe CDDT results are highly consistent

10

9

8

7

6

5

4

3


601

895

ln AUCACTH

Figure 2 The mean individual log-transformed ACTH area underthe curve (AUC) obtained in the CDDT in Cushingrsquos diseaseremission and uncured patients (119875 lt 0001) with standard errorsof the mean (SEM)

with the traditional hormonal assessments at month 18following transsphenoidal pituitary surgery


4 Discussion

The desmopressin-induced ACTH and cortisol stimulationtest has been used in CD diagnostics since the mid-90s [11ndash13] in several European and American centres Its use isbased on the confirmed presence of V3 vasopressin receptorsexpressed on the cells of themajority of pituitary corticotrophtumors [12] Desmopressin (DDAVP) is a vasopressin ana-logue that lacks an amine group at position 1 and that hasD-arginine at position 8 in place of the physiologicallyoccurring L-isomer These modifications lead to a prolongedbiological activity of the analogue and its significantly largerselectivity towards the V3 receptor in comparison with theoriginal molecule This translates into the elimination of thevasoconstriction and hypertensive effect which definitelyincreases the safety of DDAVP use compared with vaso-pressin Initially the desmopressin test was used only inthe differential diagnostics of ACTH-dependent Cushingrsquossyndrome [14 15] The test was not explicitly regarded byendocrine societies a management standard although itgained popularity especially in Italian and French centresSome literature data suggest that the desmopressin test ischaracterized by better diagnostic accuracy in differentiatingmild-to-moderate cases of Cushingrsquos disease from the so-called pseudo-Cushingrsquos states [13ndash15] in comparison to acorticoliberin test performed after a low dose dexamethasonesuppression test (4 times 05mg for 2 days) recommended bythe consensus of 2008 [1] Hence it seems that diagnosingCushingrsquos disease with moderate hypercortisolemia will con-stitute in the near future the fundamental area for diagnosticuse of the test more so as the European recommendationsand commentaries indicate purposefulness of conductingscientific research towards this direction Simultaneouslyafter years of gathering experience in the diagnostics ofCushingrsquos syndrome the DDAVP stimulation test started tobe analyzed in terms of its use in the assessment of the efficacyof surgical treatment of corticotroph pituitary adenomas [78]

In the dissertation published in 2009 Losa et al demon-strated that persistent postoperative ACTH and cortisolresponse to DDAVP in the first week after surgical treatmentincreases the probability of CD recurrence A postoperativeresponse to desmopressinwas to suggest a persistent presenceof tumor cells which are characterized by the autonomy ofACTH secretion In such cases desmopressin stimulationwould lead to a significant increase of both ACTH andcortisol secretion However themain objection raised againstthis test is the possibility of V3 receptors being present onnormal corticotroph cells although the frequency of thisphenomenon has not been explicitly determined [7] Yetthe two indisputable advantages of this test are its wideavailability and a considerably lower cost compared to thoseof the CRH test [2 10]

Our study attempted to assess the frequency and clinicalsignificance of a positive desmopressin test result performedafter surgical treatment of corticotroph pituitary adenomaWe also compared the obtained results of this test withtreatment efficacy assessment results (conducted on the basisof the previously discussed remission criteria) performed

18 months after TSS However in order to eliminate thepossibility of normal corticotroph cell stimulation by desmo-pressin a modified version of this test was used whichconsisted in administering a DDAVP dose 8 hours afterthe previous administration of 1mg of dexamethasone Thisleads to the suppression of normal pituitary corticotroph cellfunctions by a ldquolowrdquo dose of dexamethasone At that pointonly autonomous cells undergo stimulation if they are stillpresent in the pituitary gland or the parasellar structures[9] In our study the test was performed not immediatelyafter surgery as was the case in studies by Losa et al [7]but approximately 18 months after surgical treatment Thisis because it seems that directly after surgery one can assessthe functional state of the pituitary-adrenal axis on the basisof serum cortisol or urinary free cortisol levels in a 24-hoururine collection A confirmation of secondary adrenal cortexdeficiency conducted in this manner is sufficient to regard agiven patient as being in remission of CD Within the periodof 18 months from surgery patients remaining in remissiongradually regain adrenal cortex function and their serumcortisol levels increase According to our unpublished obser-vations over 80of patients in remission regain their adrenalcortex function one and a half years after the operation whichmakes it possible to decrease and finally discontinue theirhydrocortisone replacement therapy In the group presentedhere only 3 out of 15 patients in remission (20) continued torequire hydrocortisone replacement therapy (10mg dose inall 3 cases)This is precisely at this crucial time point when theresults of both the 1mg dexamethasone test (which precedesDDAVP administration) and desmopressin stimulation testprovide valuable information that helps distinguish with ahigh probability the returning of normal adrenal functionfrom an onset of the recurrence of hypercortisolemia In theformer case one should expect a decrease in serum cortisollevels to the value of lt18 120583gdL and a negative result of thedesmopressin test and in the latter one cortisol levels ofge18 120583gdL and a positive desmopressin test result These twoclinical situations cannot be initially differentiated solely onthe basis of routine serum cortisol levels or in a 24-hour urinecollection which in both cases may be within the relevantlaboratory reference ranges

Thus it seems that a positive ACTH and cortisol responseto desmopressin after the period of 15 years from surgicaltreatment may be treated as an unfavorable prognosticfeature Such results should lead to a particularly carefuland prolonged postoperative follow-up especially in light ofclinical observations indicating cases of late CD recurrenceeven after 10 years from the initially effective surgery [3 6]The important role of this test is also confirmed by the highlevel of conformity between the result of a CDDT and theassessment based on the established criteria of remissionthat include achievement of normal cortisol levels withmaintained circadian rhythm and normal suppression in the1mg dexamethasone test

The demonstrated conformity between the negative resultof the CDDT and subnormal serum cortisol levels on day1 or 2 after TSS for CD is particularly noteworthy Thus itseems that a negative test result may be an additional factorindicating CD remission


Our results indicate that maximum plasma ACTH con-centrations were achieved at the 30-minute time point ofthe test or earlier and maximum cortisol concentrationsin over 90 of patients were achieved within 90 minutesafter DDAVP administration Thus it seems that for clinicalpurposes ACTH and cortisol levels may be measured for ashorter period of time (ie for up to 90 minutes) than it hasbeen recommended by other authors namely for 120ndash180minutes [7 9]

The assessment of inconclusive test results such as thosewhich were achieved in the case of 2 patients still remainsan open question requiring further investigations (includingthose conducted in a larger group of patients with CD) Bothof our patients with inconclusive results demonstrated a fairlysignificant relative increase (Δ) inACTHwith basically lowabsolute values of this hormone as well as a lack of cortisolresponse to DDAVP stimulation Both of these patients wereyoung women aged under 25 at the onset of CD Oneof them developed recurrence of hypercortisolemia after 5years of follow-up which required another surgery (currentlythe patient is in early remission 6 months after surgery)The elevated plasma ACTH levels observed in this womanduring the test preceded both the clinical manifestations ofhypercortisolemia and a deterioration in terms of traditionalhormonal parameters The other young woman continuesher follow-up without evidence of disease recurrence Theanswer to the question of how often such patients develop arecurrence of hypercortisolemiawill definitely require a long-lasting follow-up and a larger sample size

5 Conclusion

In conclusion it should be stated that the presented ACTHand cortisol stimulation test after prior suppression of phys-iological secretion with a low dose of dexamethasone mayfacilitate an earlier diagnosis of a possible recurrence ofhypercortisolemia In diagnostically difficult cases the CDDTcan help determine a plan of further treatment [10] Thusthe test may be beneficial in diagnostically difficult cases ofCD particularly as part of postoperative assessments In abroader sense this is a reminder of the fact that dynamictesting in CD constitutes a valuable and very often necessarysupplement to routine hormonal assessments performed inbasic conditions

6 Study Strengths and Limitations

Like in other studies on CD the main limitation of our workwas a small sample size Nonetheless the low prevalence ofCD should not discourage anyone from collecting clinicaldata especially on procedures that raise doubts and havenot yet become part of standard diagnostic managementThus in accordance with the Endocrine Society ClinicalPractice Guideline and European Commentaries we evalu-ated prospectively a homogeneous group of patients froma single pituitary centre in order to collect more data onthe clinical usefulness of the desmopressin test (in the cou-pled desmopressin-dexamethasone version) in postoperative

follow-up of patients after surgical treatment of CD Furtherlimitation to this study was a relatively short follow-upperiod due in part to the prospective character of this studyAlso the presented version of the test has been used fora relatively short time not allowing us to fully determinehow to establish the positive and negative test result valuesPerhaps this should be based on only one of the evaluatedparametersmdasheither ACTH or cortisol The advantage ofACTH is produced by the same cells that undergo stimulationwith desmopressin Conversely the advantage of cortisol isgreater stability and higher repetition of results

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

This work and paper preparation was supported by thegrant of Military Institute of Medicine Warsaw Poland (no18807(258)2013)

References

[1] L K Nieman BM K Biller JW Findling et al ldquoThe diagnosisof Cushingrsquos syndrome an endocrine society clinical practiceguidelinerdquo Journal of Clinical Endocrinology and Metabolismvol 93 no 5 pp 1526ndash1540 2008

[2] L Guignat and J Bertherat ldquoThe diagnosis of Cushingrsquossyndrome an Endocrine Society clinical practice guidelinecommentary from a European perspectiverdquo European Journalof Endocrinology vol 163 no 1 pp 9ndash13 2010

[3] G Zada ldquoDiagnosis and multimodality management of Cush-ingrsquos disease a practical reviewrdquo International Journal ofEndocrinology vol 2013 Article ID 893781 7 pages 2013

[4] P Witek G Zielinski M Maksymowicz and G KaminskildquoCushingrsquos diseasemdashassessing the efficacy of transsphenoidalsurgeryrdquo Endokrynologia Polska vol 63 no 5 pp 398ndash4042012

[5] S-C Tung P-WWang R-T Liu et al ldquoClinical characteristicsof endogenous Cushingrsquos syndrome at a medical center inSouthern Taiwanrdquo International Journal of Endocrinology vol2013 Article ID 685375 7 pages 2013

[6] C G Patil D M Prevedello S P Lad et al ldquoLate recurrencesof Cushingrsquos disease after initial successful transsphenoidalsurgeryrdquo Journal of Clinical Endocrinology and Metabolism vol93 no 2 pp 358ndash362 2008

[7] M Losa R Bianchi R Barzaghi M Giovanelli and P MortinildquoPersistent adrenocorticotropin response to desmopressin inthe early postoperative period predicts recurrence of Cushingrsquosdiseaserdquo Journal of Clinical Endocrinology and Metabolism vol94 no 9 pp 3322ndash3328 2009

[8] M Losa P Mortini S Dylgjeri et al ldquoDesmopressin stimu-lation test before and after pituitary surgery in patients withCushingrsquos diseaserdquo Clinical Endocrinology vol 55 no 1 pp 61ndash68 2001


[9] F Castinetti M Martinie I Morange et al ldquoA combineddexamethasone desmopressin test as an early marker of post-surgical recurrence in Cushingrsquos diseaserdquoThe Journal of ClinicalEndocrinology ampMetabolism vol 94 no 6 pp 1897ndash1903 2009

[10] P Witek W Zgliczynski G Zielinski and W Jeske ldquoThe roleof combined low-dose dexamethasone suppression test anddesmopressin stimulation test in the diagnosis of persistentCushingrsquos disease Case reportrdquo Endokrynologia Polska vol 61no 3 pp 312ndash317 2010

[11] P Colombo E Passini T Re G Faglia and B Ambrosi ldquoEffectof desmopressin on ACTH and cortisol secretion in states ofACTH excessrdquo Clinical Endocrinology vol 46 no 6 pp 661ndash668 1997

[12] Y de Keyzer P Rene C Beldjord F Lenne and X BertagnaldquoOverexpression of vasopressin (V3) and corticotrophin-releasing hormone receptor genes in corticotroph tumoursrdquoClinical Endocrinology vol 49 no 4 pp 475ndash482 1998

[13] MMoro P PutignanoM Losa C Invitti CMaraschini and FCavagnini ldquoThe desmopressin test in the differential diagnosisbetween Cushingrsquos disease and pseudo-Cushing statesrdquo Journalof Clinical Endocrinology and Metabolism vol 85 no 10 pp3569ndash3574 2000

[14] F P Giraldi R Pivonello A G Ambrogio et al ldquoThe dexam-ethasone-suppressed corticotropin-releasing hormone stimula-tion test and the desmopressin test to distinguish Cushingrsquos syn-drome from pseudo-Cushingrsquos statesrdquo Clinical Endocrinologyvol 66 no 2 pp 251ndash257 2007

[15] G Tirabassi E Faloia R Papa G Furlani M Boscaro andG Arnaldi ldquoUse of the desmopressin test in the differentialdiagnosis of pseudo-cushing state from cushingrsquos diseaserdquo TheJournal of Clinical Endocrinology amp Metabolism vol 95 no 3pp 1115ndash1122 2010

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

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EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

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PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


The aim of this study was to conduct a prospectiveevaluation of the role and usefulness of the combined dex-amethasone desmopressin test in postoperative assessmentsof transsphenoidal surgery effectiveness in CD patients in asingle pituitary center

2 Material and Methods

21 Patient Population The study population comprised 28consecutive patients with Cushingrsquos disease (22 women and6 men F M ratio 37 1) hospitalized and operated in theMilitary Institute of Medicine in Warsaw between 2010 and2012 The mean age was 361 plusmn 135 years (168ndash576) Aftertheir confirmed diagnosis of Cushingrsquos disease the patientswere operated on by the same neurosurgeon using the samesurgical protocol

All patients were informed about the aims and methodsof the study and they signed the informed consentThe studyprotocol was approved by the local ethics committee

22 Preoperative Endocrine Evaluation All patients under-went a standard clinical evaluation The diagnosis of ACTH-dependent Cushingrsquos syndrome (CS)wasmade based on clin-ical signs and standard hormonal criteria increased urinaryfree cortisol (UFC) increased morning serum cortisol levelat 800 loss of cortisol circadian rhythm (the serum cortisollevel above 75 120583gdL in the late-night hours (2200ndash0000))and increased or detectable level of plasma ACTH at 800and the failure of serum cortisol to suppress to less thanor equal to 18 120583gdL during the low dose dexamethasonesuppression test (LDDST 05mg qid for 48 hours) Thepituitary etiology of CS was confirmed based on serumcortisol or UFC suppression greater than 50 with thehigh dose dexamethasone suppression test (HDDST 2mgqid for 48 h) and positive pituitary MRI In the case ofinconclusive hormonal assessments and pituitary imagingthe diagnosis of Cushingrsquos diseasewas confirmed by a positiveresult of a stimulation test with intravenous CRH injection(100 120583g) andor results of inferior petrosal sinus sampling(IPSS)

23 Preoperative MRI Prior to neurosurgical treatmentall patients underwent high-resolution magnetic resonanceimaging of the pituitary-hypothalamic region (SIEMENSSymphony 2004 15 Tesla) performed before and after intra-venous injection of gadolinium (Gd-DTPA) The presencesize and position of any focal lesions were recorded Ahypodense lesion visualized after contrast administrationwasconsidered to be indicative of pituitary adenoma Microade-noma was defined as a pituitary tumor with a diameter of lessthan 1 cm whereas macroadenoma was defined as a pituitarytumor with at least one diameter of more than 1 cm

24 Surgical Procedure A microsurgical transseptal trans-sphenoidal approach was used for resection of ACTH-secreting pituitary adenomas The pituitary gland was care-fully examined regardless of MRI findings Selective ade-nomectomy was performed in all cases of MRI-visualized

pituitary adenomas Where MRI findings were inconclusiveor no tumor was evident a series of vertical and horizontalincisions of the pituitary gland were performed with all tissuedeemed abnormal removed and submitted for pathologicalexamination

25 Histopathological and Immunohistochemical AssessmentSurgical specimens were collected for histopathological anal-ysis (standard hematoxylin and eosin staining) and immuno-histochemical staining for pituitary hormones (kits withpolyclonal antibodies Dako Denmark) The result of ahistopathological assessment was considered to be positiveif the presence of corticotroph adenoma as well as positiveACTH-staining was confirmed in histological and immuno-histochemical examinations The result was treated as nega-tive if there was no histopathological evidence of corticotrophadenoma and ACTH-staining was negative

26 Postoperative Hormonal Evaluation and Criteria of CureBlood samples for serum cortisol measurements were col-lected from all patients at 600 on the first postoperativeday Glucocorticoids were not administered in the periop-erative or in the early postoperative period Hydrocortisonereplacement therapy was started after biochemical confir-mation of hypocortisolemia or the development of clinicalmanifestations of adrenal insufficiency The standard dose ofhydrocortisone (20mg in the morning and 10mg at 1500)was introduced when necessary and continued until the nexthormonal evaluation

Following the surgical procedure all patients weresubjected to further postoperative evaluations Subsequentreassessments were performed at 6 weeks and at 3 6 1218 and 24 months after surgery and once yearly thereafterPatients on hydrocortisone replacement therapy had theircortisol measurements taken 48 hours after the last admin-istered dose We used the following criteria for sustainedremission biochemical evidence of eucortisolemia (morningserum cortisol in the 5ndash25 120583gdL range) preserved circadianrhythm of serum cortisol (the late-night serum cortisol levelle75 120583gdL) and the ability of serum cortisol to suppressto less than or equal to 18 120583gdL after a 1mg overnightdexamethasone suppression test (ODST)

27 Combined Dexamethasone Desmopressin Testing Thecombined dexamethasone desmopressin test (CDDT) wasconducted in two stages After the informed consent had beensigned each patient received 1mg of oral dexamethasoneat midnight In patients who had received hydrocortisonereplacement therapy it was discontinued 48 hours prior tothe test A 10 120583g of desmopressinmdashMinirin (Ferring GmbHGermany)mdashwas administered as a slow intravenous bolus8 hours after dexamethasone administration Blood samplesfor testing ACTH and cortisol levels were collected beforeand 15 30 60 90 and 120 minutes after desmopressinadministration

Apositive test result which indicates persistent impropercorticotroph secretory response to desmopressin requiresplasma ACTH increase by at least 30 and serum cortisol







3 Results




































10

9

8

7

6

5

4

3

509

759


ln AUCcortisol



10

9

8

7

6

5

4

3


601

895

ln AUCACTH




4 Discussion










5 Conclusion







Acknowledgment


References






















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















3 Results




































10

9

8

7

6

5

4

3

509

759


ln AUCcortisol



10

9

8

7

6

5

4

3


601

895

ln AUCACTH




4 Discussion










5 Conclusion







Acknowledgment


References






















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of









































10

9

8

7

6

5

4

3

509

759


ln AUCcortisol



10

9

8

7

6

5

4

3


601

895

ln AUCACTH




4 Discussion










5 Conclusion







Acknowledgment


References






















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















4 Discussion










5 Conclusion







Acknowledgment


References






















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















5 Conclusion







Acknowledgment


References






















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















clinical study cushing s disease: the relevance of a

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