Differential diagnosis of ACTH-dependent CS is challenging and to date a single best approach in the diagnostic work-up of these patients does not exist.
Whereas the usefulness of stimulatory and suppression tests is widely accepted, their role to the light of positive MRI (pituitary adenoma < or > 6 mm) or negative findings is still a matter of debate. In the latter case, although BIPSS still represents the gold-standard procedure for differential diagnosis regardless the results of dynamic tests [7, 18], different clinical approaches and opinions are reported in the literature.
In a recent opinion statement by members of the Italian Society of Endocrinology, Italian Society of Neurosurgery and Italian Society of Neuroradiology that summarizes different strategies adopted in the prescription of BIPSS , the authors report two studies in which BIPSS did not show any influence on neurosurgical remission rates. In the first one, Bochicchio and coll. retrospectively analyzed data from 668 patients affected by CD and described that in 98 subjects who underwent BIPSS, surgical failure was similar to patients who did not ; however, in this cohort CRH and TRH tests but not HDDST, were performed and selection criteria for BIPSS were not clearly reported. In the second one, Jehle and coll. performed a retrospective analysis of 193 patients with ACTH-dependent CS ; also in this case, BIPSS did not affect remission rate after TSS as far as recurrence and long-term remission rates. The procedure was reserved to patients with equivocal scan and/or biochemical tests; however, biochemical evaluation consisted of ACTH and UFC levels, while CRH test was not performed and data about HDDST were lacking in all but six patients.
In a subsequent review about the role of BIPSS in CS, Zampetti et al.  suggested that, on the basis of authors’ experience, BIPSS should not be performed in patients with positive response to CRH test (defined as increase > 50% in ACTH and > 30% in cortisol), particularly if a consistent suppression to HDDST is present, independently of MRI findings. This opinion was finally remarked by Losa et al.  which pointed out CRH test as the main factor in providing indication to BIPSS.
In this area of controversy, we performed a retrospective analysis on 148 patients with CD and 26 patients with EAS aiming to evaluate the role non-invasive tests in the diagnostic work-up, with secondary focus on the need of BIPSS in CD patients with inconclusive neuroradiological examination. In all 148 patients of our cohort, the diagnosis of CD was confirmed by biochemical remission after TSS, histology and/or > 6 months post-surgical hypoadrenalism.
In agreement with previous data, our results confirm that CRH test and HDDST have high accuracy in differential diagnosis of ACTH-dependent CS [8, 9, 27]. As a whole, a positive response was observed in 89.4% and 91.4% of patients with CD, and in 3.8% and 23.1% of patients with EAS, respectively. More importantly, the combination of concordant positive responses to CRH test and HDDST reaches 100% specificity and PPV, thus allowing the diagnosis of CD irrespective of MRI findings. Otherwise, a single-test approach is not able to reach a specificity of 100%. The same performance is maintained in the subgroup of patients with negative MRI or with a microadenoma < 6 mm. Furthermore, in this subgroup, a negative response to both CRH test and HDDST is sufficient to make the diagnosis of EAS.
Interestingly, in CD patients, the response rate to CRH test, as far as ACTH and cortisol percentage increase, were significantly higher in patients with microadenomas or negative imaging in respect to those with macroadenomas. A similar observation was recently reported in a group of 149 CD patients where macroadenomas tended to show a lower increase of ACTH after CRH compared to microadenomas . As a negative correlation between baseline secretion and ACTH and cortisol responses to CRH in CD patients has been described , suggesting in this context a different degree of negative feedback impairment at the pituitary level, the finding of higher baseline ACTH levels in our patients may represent the most likely explanation for this observation.
Accordingly, the highest rate of false negative responses to dynamic tests were observed in patients with macroadenomas, in which a false negative result to both CRH and HDDST was recorded in four cases; nevertheless, in this condition BIPSS is already overlooked due to the low pretest probability of the co-existence of a pituitary macroadenoma and an ectopic CS.
The role of DDAVP test in differential diagnosis of ACTH-dependent CS is still controversial and a high frequency of false positive results in patients with EAS has been reported . However, in a recent work including 167 patients with CD and 27 patients with EAS, the positive response to both CRH and DDAVP test showed a positive predictive value of 100% for CD in patients with negative MRI and negative computed tomography scan . In our study, similarly to CRH test and HDDST, also the combination of positive responses to both CRH and DDAVP tests reaches a specificity and PPV of 100% for the diagnosis of CD. However, DDAVP test presents low sensitivity and specificity, thus resulting in a high prevalence of false negative and false positive results as well as a concordance rate significantly lower than that observed for CRH test and HDDST in patients with negative MRI or with a microadenoma < 6 mm. In addition, in four of these patients we recorded a concordant negative response to CRH and DDAVP tests that might have resulted in misdiagnosis. Therefore, our data indicate that DDAVP test may represent a valid alternative, in particular when discordant results arise from other dynamic tests, but CRH test, HDDST and their combination perform better and reduce the need to perform BIPSS.
On the other hand, it is well recognized that DDAVP may have an important role in the post-surgical follow-up of CD patients, as the persistence or reappearance of a positive response may precede the clinical recurrence of disease [21, 22, 33,34,35,36,37,38].
In our series, BIPSS confirmed the diagnosis of CD in 28 out of 30 patients who underwent this procedure. Two negative cases included one patient with a pituitary adenoma sized between 6 and 10 mm but discordant CRH test and HDDST and another one with negative imaging and concordant tests. Notably, in the latter case, a borderline central/periphery ratio of 2.91 was recorded. Nevertheless, diagnosis of CD was subsequently proven by remission after neurosurgery, suggesting that BIPSS returned a false negative result in both patients. The proportion of false negative we observed is in line with previous literature data reporting a prevalence of 3–19%, possibly related to anatomical or biochemical variations of disease [14, 17, 27, 30, 39, 40]. Furthermore, BIPSS is burdened by possible complications. In particular, minor adverse events (i.e., groin hematoma, tinnitus, otalgia) have been reported in about 4% of patients, while severe complications (i.e., brainstem infarction, subarachnoid haemorrhage, pulmonary and deep venous thrombosis) are expected in less than 1% of cases [27, 30]. As reported above, in our series one patient died 24 h after BIPSS due to cardiac rupture, while no complications in the other subjects were recorded. Although our fatal event was unlikely related to the procedure and complications are rare, all these observations point out the need for an accurate selection of patients referred to BIPSS.
Following the results of diagnostic performance analysis, in those patients with concordant positive responses to CRH test and HDDST but inconclusive neuroradiological findings (i.e., negative imaging or pituitary adenoma < 6 mm), the execution of BIPSS did not improve surgical outcome. Then, our data do not support the routine use of BIPSS in this subgroup of CD patients, in whom BIPSS could have been avoided in 22 out of 29 subjects. In this setting, contrarily to what the current guidelines propose [7, 13, 18, 19], CRH test and HDDST seems to be sufficient to confirm the diagnosis of CD and to provide indication to pituitary surgery. Similarly, a negative response to both tests pointed toward EAS diagnosis; in this circumstance BIPSS can be avoided too. Indeed, the present study does not propose to remove BIPSS from the diagnostic work-up of ACTH-dependent CS diagnosis, but to restrict its use when really necessary.
Our study has some limitations: first, its retrospective nature, leading in particular to an inhomogeneous selection of patients referred to BIPSS. Second, our data do not allow to draw conclusions about patients with intermediate pituitary lesion between 6 and 10 mm. Although our approach was to avoid BIPSS even in case of discordant results, except in the presence of clinical features suggestive for ectopic CS (rapid onset, hypokalemia, advanced age), these cases can still represent matter of debate.
On the other side, the strength is represented by the comprehensive and punctual biochemical and diagnostic characterization of patients which in our view makes our results very reliable.
In conclusion, our study confirms that CRH test, DDAVP test and HDDST have high accuracy in the differential diagnosis of ACTH-dependent CS. In particular, the combination of CRH test and HDDST allows to achieve the best performance in terms of sensitivity and specificity. In patients with negative MRI or with a microadenoma < 6 mm, the presence of concordant positive response to CRH test and HDDST or to CRH test and DDAVP test seems to be sufficient to establish the diagnosis of CD. In this subgroup of patients, BIPSS should be therefore reserved for those cases with discordant tests.