DIAGNOSIS AND PROGNOSIS OF ADRENAL BENIGN AND MALIGNANT TUMORS: NEW INSIGHTS FROM A COMPUTERIZED OPERATOR-INDEPENDENT MORPHOMETRIC MODEL AND PROTEOMIC EXPRESSION

Background: High mitotic index, high nuclear grade and reticulin distruption are part of representative hallmarks of adrenocortical cancer (ACC). A characteristic neutrophil/T-lymphocytes infiltrate ratio has been often implicated in carcinogenesis, progression and clinical outcome of several cancer types. However, its role in adrenal cortical tumors is unclear. Histology-based diagnosis may also suffer of a moment of subjectivity due to inter- and intra-observer variations. Proteomic studies of malignant tumors represent the future both for possible diagnostic and prognostic implication; whole proteome analysis of adrenocortical tumors from fresh tissues may represent the lacking piece of puzzling management of this tumor. Aim: to assess by computerized morphometry morphological features, vascular, inflammatory, reticulin and proliferative index pattern in adrenocortical adenomas (ACAs) and carcinomas and to assess proteomic profiles from adrenocortical tumors fresh tissues. Methods: A single Institution series of 11 ACAs and 18 ACCs samples was analyzed using a Kontron-Zeiss KS400 image analyzer. Four consecutive sections 4 \ub5m thick were obtained with a total of 250\u2013300 HPF examined for each case. Immunohistochemistry for Ki67, reticulin and CD8/CD15 was obtained. To minimize subjectivity, particularly relevant when quantitative results are expected, we generated a morphometric model based on analysis of volume fractions occupied by Ki67 positive and negative cells (nuclei, cytoplasm) and inflammatory compartments (CD15+ granulocytes, CD8+ lymphocytes) and reticulin framework surface. Lastly, the assessment of Ki-67 by computerized morphometry was compared with pathologist\u2019s evaluation. After sample preparation protocol of 7 ACCs, 5 ACAs and 5 normal adrenal tissue samples, difference In Gel Electrophoresis (DIGE) and following the protein spots individuation and isolation, Mass Spectrometry, were performed to allow protein identification. Results: The volume fraction of Ki-67 positive cells was highest in ACC. The volume fraction of nuclei in unit volume and the nuclear/cytoplasmic ratio in both Ki-67 negative cells and Ki-67 positive cells were prominent in ACC. The surface fraction of reticulin was considerably lower in ACC. Moreover, when comparing morphometric analysis of Ki67+ cells to pathologist\u2019s scores, the data of the point grid analysis revealed significantly lower values compared to conventional histopathology. These values, once statistically analyzed, demonstrated that our morphometric model could improve the sensitivity and specificity of Ki-67 evaluation in ACCs and ACAs (reaching 94% of sensitivity, 100% of specificity) and also that it cold contribute to a better prognosis definition. Proteomics individuated 62% overexpressed proteins in ACCs with respect to ACAs: among them vimentin and vitamin D-binding protein resulted the most varied (3.2 and 3-fold more expressed in ACCs than in ACAs respectively). On the other hand the remaining 38% of proteins resulted under-expressed in ACCs with regard to ACAs, being cathepsin D and aldose reductase both 3-fold less expressed in ACCs than in ACAs. The protein profile of ACCs versus normal adrenal tissue was similar (although with slight differences in terms of fold variation) to that of ACCs versus ACAs; nonetheless a varied new protein (lactate dehydrogenase, 1.8 fold increase in ACCs) with a possible role in tumorigenesis and tumor progression, was detected. Conclusions: Our computerized morphometric model is simple, lacks observer or subjective bias and can be used to supplement objective methods to achieve precise and reader-independent quantification of morphological characteristics and histological biomarkers of adrenocortical tumors. We speculate that the assessment of inflammatory infiltrate may found a place in the diagnostic algorithm of adrenal benign and malignant tumors. The promising preliminary results obtained by the proteomic study of ACCs and ACAs could contribute to the identification of new histological biomarkers. These data, once integrated into a complex algorithm including histological assessment, morphometric analysis and clinical data evaluation would easily contribute to create a prognostic stratification of ACCs with clear advantages for the clinical management of the disease

Management of occult adrenocorticotropin-secreting bronchial carcinoids : limits of endocrine testing and imaging techniques

The differential diagnosis and the identification of the source of ACTH in occult ectopic Cushing's syndrome due to a bronchial carcinoid still represents a challenge for the endocrinologist. We report our experience in six patients with occult bronchial carcinoid in whom extensive hormonal, imaging, and scintigraphic evaluation was performed. All patients presented with hypercortisolism associated with high plasma ACTH values. The CRH test and high dose dexamethasone suppression test suggested an ectopic source of ACTH in three of six patients. During bilateral inferior petrosal sinus sampling, none of the patients showed a central to peripheral ACTH gradient. At the time of diagnosis, none of the patients had radiological evidence of the ectopic source of ACTH, whereas pentetreotide scintigraphy identified the lesion in two of four patients. Finally, a chest computed tomography scan revealed the presence of a bronchial lesion in all patients, and pentetreotide scintigraphy identified four of six lesions. In all patients a bronchial carcinoid was found and removed. In one patient with scintigraphic evidence of residual disease after two operations, radioguided surgery, using a hand-held \u3b3 probe after iv administration of radiolabeled pentetreotide, was performed; this allowed detection and removal of residual multiple mediastinal lymph node metastases. In conclusion, our data show that there is not a single endocrine test or imaging procedure accurate enough to diagnose and localize occult ectopic ACTH-secreting bronchial carcinoids. Radioguided surgery appears to be promising in the presence of multiple tumor foci and previous incomplete removal of the tumor

Spontaneous recovery of bone mass after cure of endogenous hypercortisolism

Patients with Cushing's syndrome (CS) develop osteopenia-osteoporosis. The present study evaluates the recovery of bone mass within 2 years after remission of hypercortisolism and in long term follow up, an issue rarely addressed. Twenty patients (6M, 14F, 3 post-menopausal, 15-64 years old), 15 with Cushing's disease, 2 with ectopic ACTH syndrome, 3 with ACTH-independent CS were studied. BMD, T and Z scores at lumbar spine and proximal femur were assessed by dual-energy X-ray absorptiometry before and 7-33 months after treatment of hypercortisolism. Five patients were treated with bisphosphonates. Four patients had hypogonadism and 4 GH-deficiency. At baseline all patients showed osteopenia/osteoporosis and the spine appeared more damaged than the femur; femur BMD was positively related with body mass index (BMI). No correlations were observed between spine and femur bone parameters and duration of disease or severity of hypercortisolism. Bone parameters did not differ in patients with or without GH or other pituitary deficiencies. After cure of hypercortisolism a significant improvement in spine BMD, Z and T scores and in femur Z and T scores was observed with normalization in 3 patients; there was no significant difference in percent improvement between femur and spine. The increase in bone parameters at spine and femur was independent from values at baseline. The percent increase in spine T and Z scores was positively related with time elapsed since cure. Bisphosphonates did not influence the recovery of bone mineralization. In long term follow up, after a median period of 7 years a further improvement in bone density was observed in 100% of patients at spine and in 9/11 at femur, although 8/11 patients still had femoral and/or vertebral T score in the range of osteopenia/osteoporosis. Spontaneous improvement of osteoporosis after cure of hypercortisolism occurs both at spine and femur, is independent from basal conditions and not affected by bisphosphonates. The improvement at spine depends on time since cure

Adrenocortical Cancer: Use of New ENSAT Staging System and Re-Evaluation of Old and New Markers of Prognosis

A modification of the TNM classification (WHO-UICC 2004) for adrenocortical cancer (ACC) has been recently proposed in order to improve the prognostic value as to the disease-free and disease-specific survival.(1) So far an increasing number of molecular markers has been proposed for early detection, confirmation of malignancy and/or outcome prediction, but none of them is considered as reliable as the Weiss score morphological system.(2,3) Aim: to compare the prognostic value of WHO-UICC 2004 and new ENSAT TNM staging classification in a single Institution ACC series as to the disease-free and disease-specific survival and to compare these findings with tumor Weiss score and ki67 expression. Methods: Clinical and pathological data of 26 pts (19F, 7M, median age 52 yrs, range 25-78) surgically treated for ACC, were retrospectively reviewed; the follow up period was 26 months (median, range 6-192). ACC staging was performed according to both WHO-UICC 2004 and ENSAT classification, tumor malignancy was assessed according to Weiss system. A multivariate analysis of disease-free and overall survival in ACC according to Weiss score and ki67 (IHC, monoclonal Mib1 Ab) was performed. Results: 70% of tumors were hypersecreting ; mean size was 10,7cm (range 50-2500), mean weight was 512g (range 50-2500). WHO-UICC identified 2 stage I, 11 stage II, 4 stage III and 9 stage IV disease (Kaplan-Meyer, log-rank test: p=0,0004). Two pts with stage IV WHO-UICC and good prognosis switched to stage III ENSAT (Kaplan-Meyer, log-rank test: p=0,0002). In our series ki67>7% (sensitivity 85,7%, specificity 62,5%, ROC analysis) and Weiss score>4 (sensitivity 100%, specificity 50%) were the best cut-off values suggestive for a poor prognosis (Kaplan-Meyer, p=0,06 and p=0,04 respectively); taken together the two risk factors showed a significant effect on survival probability (Cox-regression, p= 0,04). Ki67>7% and Weiss score>4 showed a slight correlation also with disease-free survival (Kaplan-Meyer, p=0,06 and p=0,04 respectively); when both risk factors were concomitantly present, the correlation with probability of disease recurrence was higher (Cox-regression, p=0,02). Conclusions: The outcome of pts with ACC is strictly related to the stage of the disease, being new ENSAT proposal a valuable tool to improve the power of the stage-related prognostic value. Ki67 expression and Weiss score combined may play a role of interest, but more reliable molecular markers are needed

Quantitative Assessment By Computerized Morphometry of Ki-67 and Intratumoral Inflammatory Infiltrate in Benign and Malignant Adrenocortical Tumors

High mitotic index and high nuclear grade are part of representative hallmarks of adrenocortical cancer (ACC), but the analysis of these parameters is known to be operator-dependent. A characteristic neutrophil/T-lymphocytes infiltrate ratio has been often implicated in carcinogenesis, progression and clinical outcome of several cancer types. However, its role in adrenal cortical tumors is unclear. Aim: to assess by computerized morphometry morphological features, vascular and inflammatory pattern in adrenocortical adenomas (ACAs) and carcinomas. Methods: A single Institution series of 11 ACAs and 18 ACCs samples was analyzed using a Kontron-Zeiss KS400 image analyzer. Four consecutive sections 4 \ub5m thick were obtained with a total of 250\u2013300 HPF examined for each case. Immunohistochemistry for Ki67 and CD8/CD15 was obtained to assess proliferation index and inflammatory infiltrate respectively. To minimize subjectivity, particularly relevant when quantitative results are expected, we generated a morphometric model based on analysis of volume fractions occupied by Ki67 positive and negative cells (nuclei, cytoplasm) and inflammatory compartments (CD15+ granulocytes, CD8+ lymphocytes). Lastly, the assessment of Ki-67 by computerized morphometry was compared with pathologist\u2019s evaluation. Results: volume fraction of Ki-67+ cells was higher in ACCs (ACC .11951, ACA .06637; p<0.001); nuclei volume fraction resulted higher in ACCs, in both Ki-67- (ACC .11951, ACA .06637; p<0.001) and Ki-67+ cells (ACC .01293, ACA .00104; p<0.001). Nuclear/cytoplasmic ratio was higher in ACCs, both Ki-67- (ACC .20535, ACA .09260; p<0.001) and Ki-67+ cells (ACC .66141, ACA .27281; p<0.001). Volume fractions of CD15+ (ACC .00312, ACA .00098; p<0.001) and CD8+ cells (ACC .00731, ACA .00356; p<0.05) were also significantly higher in ACCs. Moreover, when comparing morphometric analysis of Ki67+ cells to pathologist\u2019s scores, the data of the point grid analysis revealed significantly lower values compared to conventional histopathology. Conclusions: Our computerized morphometric model is simple, repeatable (lacking observer bias) and flexible, as it can be upgraded to include newly described histological or immunohistochemical features. This method could be integrated into a classification tool to complement conventional histological analysis to achieve quantification of morphological characteristics and histological biomarkers of adrenocortical tumors. In our experience, nuclear/cytoplasmic ratio differs mostly between ACCs and ACAs, both in Ki-67+ and Ki-67- cells. We speculate that neutrophils may play a role in ACC milieu and that the quantitative assessment of inflammatory infiltrate may find a place in the diagnostic algorithm of adrenal benign and malignant tumors

Analysis of histological and immunohistochemical patterns of benign and malignant adrenocortical tumors by computerized morphometry

Diagnosis of benign and purely localized malignant adrenocortical lesions is still a complex issue. More-over, histology-based diagnosis may suffer of a moment of subjectivity due to inter- and intra-individualvariations. The aim of the present study was to assess, by computerized morphometry, the morphologicalfeatures in benign and malignant adrenocortical neoplasms.Eleven adrenocortical adenomas (ACA) were compared with 18 adrenocortical cancers (ACC). Allspecimens were stained with H&E, cellular proliferation marker Ki-67 and reticulin. We generated amorphometric model based on the analysis of volume fractions occupied by Ki-67 positive and nega-tive cells (nuclei and cytoplasm), vascular and inflammatory compartment; we also analyzed the surfacefraction occupied by reticulin. We compared the quantitative data of Ki-67 obtained by morphometrywith the quantification resulting from pathologist\u2019s visual reading.The volume fraction of Ki-67 positive cells in ACCs was higher than in ACAs. The volume fraction ofnuclei in unit volume and the nuclear/cytoplasmic ratio in both Ki-67 negative cells and Ki-67 positivecells were prominent in ACCs. The surface fraction of reticulin was considerably lower in ACCs.Our computerized morphometric model is simple, reproducible and can be used by the pathologist inthe histological workup of adrenocortical tumors to achieve precise and reader-independent quantifica-tion of several morphological characteristics of adrenocortical tumors

A Computerized, Operator-Independent Morphometric Model for the Histological Assessment of Adrenal Neoplasia: Preliminary Data

Specific biomarkers for diagnosis and prognosis of adrenocortical carcinomas (ACCs) are still lacking.(1) The proliferation marker Ki-67 is under evaluation for validation as a reliable IHC marker of malignancy for ACCs.(2) Although a well defined diagnostic cutoff has not been demonstrated yet, Ki-67 staining between 5-7% is thought to be a feature of AT malignant behavior.(3) As for the morphological markers currently used in the determination of AT malignancy, also for Ki-67 an interobserver variability is frequently reported. The aim of this pilot study was to determine, with an operator independent and repeatable technique, the proliferative activity and cell morphology of AT histological samples; a preliminary comparison with visual perception was also attempted. METHODS IHC study of 14 ACCs and 7 adrenal adenomas (AAs) samples was done (Rabbit anti-human Ki-67 monoclonal Ab, Clone SP6, 1:400); two pathologists performed a blinded examination with a consensus determination and according to Weiss score. We generated a computerized morphometric model on AT slices to evaluate the volume fractions occupied by nuclei (nVF) and cytoplasm (cVF) of both Ki-67(+)/Ki-67(-) neoplastic cells, and by inflammatory infiltrate. All morphometric variables were obtained by a computerized image analyzer (Kontron-Zeiss KS400) with a color camera attached to a light microscope (40x objective) for microscopic fields examination. More than 200 fields were systematically selected and examined by an automatically controlled procedure to assure unbiased sampling. The method of point counting by using a counting frame was used to determine the relative volume proportion of the investigated structures. Statistics: t-test. RESULTS nVF (p<0.0001), cVF (p<0.0001), nuclear/cytoplasmic ratio (p<0.0001) were the most discriminatory features between ACCs/AAs. Moreover nVF of Ki-67(+) cells was more prominent in ACCs than in AAs (p<0.0001) but in some cases an overlapping was still evident. When compared with computerized analysis, Ki-67 pathologist's evaluation in ACCs showed a marked overestimation and a wide range (average of 9,9% vs 1,4%, range 1-25 % and 0,2-4,2% respectively; p=0,00036). CONCLUSIONS Based on these preliminary data our computerized morphometric method could be considered in the future as an helpful tool for pathologists in the histological assessment of ACCs/AAs, particularly minimizing subjectivity and possibly integrating the actual morphologic Weiss criteria

The role of chromogranin A in the management of patients with phaeochromocytoma

Objective: Chromogranin A (CgA) is the most accurate general marker of neuroendocrine tumours. Supranormal CgA concentrations have been recorded in patients with tumours of neuroectodermal origin such as phaeochromocytoma and paraganglioma. Design: The present study was performed to assess the role of CgA determination in the management of patients with phaeochromocytoma, in comparison with urinary catecholamines and their metabolites. Patients: The patients studied included 22 cases with phaeochromocytoma at initial presentation or at relapse some years after surgical cure or during follow-up of a malignant phaeochromocytoma. Seventeen patients were evaluated before and after surgical removal of phaeochromocytoma. To assess the specificity of the hormonal parameters, 20 subjects were enrolled as controls; they were from a group of patients referred to our observation for possible phaeochromocytoma and who were subsequently proven not to have the disease. Results: Urinary epinephrine and norepinephrine were supranormal in 82% and 77% of patients, respectively. Urinary metanephrines and normetanephrines were supranormal in 84% and 89% of patients, respectively. The combination of urinary metanephrine and normetanephrine had a sensitivity of 100% in identifying a phaeochromocytoma. CgA was supranormal in 91% of patients. Combining the results of CgA and urinary catecholamines (epinephrine and norepinephrine), the sensitivity for diagnosis of phaeochromocytoma was 100%. Urinary catecholamines, metabolites (metanephrine and normetanephrine) and CgA levels in patients with malignant phaeochromocytoma did not differ significantly from those of patients with benign lesions. In most cases, CgA normalized after surgery. Conclusions: Our results indicate that CgA is a good marker of phaeochromocytoma; measurement of CgA could have a role in the follow-up of patients operated on for phaeochromocytoma

High expression of dopamine receptor subtype 2 in a large series of neuroendocrine tumors

Aim: To evaluate by immumohistochemistry the presence of DR subtype 2 (D2R) in well differentiated NETs of different sites and in normal islet cells. Background: Recent data in vitro and in vivo support that dopaminergic drugs might exert an inhibitory effect on hormone secretion and, possibly, on tumor growth in neuroendocrine tumors (NET)s. Their potential therapeutic role needs the demonstration of dopamine receptors (DR) in tumor cells. Little is known on the expression of DR in NETs. Results: 85% of samples (100% of bronchial carcinoids and 93% of islet cell tumors) showed positivity for D2R; intensity of immunoreaction in NETs was similar or higher than in pituitary (54% and respectively 31% of cases). D2R positivity in more than 70% of tumor cells was observed in 46% of samples. Same intensity of D2R-immunoreactivity was found in pituitary and normal islet cells. No differences in D2R expression were recorded on considering tumor grading, size, proliferative activity, presence of metastases, endocrine activity and gender. A significant difference (62.5% vs 96.4%, p = 0.039) was observed in the prevalence of D2R expression between patients with more aggressive tumors and patients without recurrence/progression of disease during follow-up. Methods: 46 NET samples from 44 patients and normal endocrine pancreatic tissue were studied. D2R-staining was performed on NETs and compared with six non-secreting pituitary adenomas and related to clinical-pathological data. Conclusion: The present data demonstrate a high expression of D2R in NETs; this finding is of clinical relevance in view of the potential role of dopaminergic drugs in inhibiting secretion and/or cell proliferation in NETs. \ua92008 Landes Bioscience

Use of radioguided surgery with [111In]-pentetreotide in the management of an ACTH-secreting bronchial carcinoid causing ectopic Cushing&apos;s syndrome

Intraoperative [111In]-pentetreotide scintigraphy with a hand-held gamma detector probe has recently been proposed to increase the intraoperative detection rate of small neuroendocrine tumors and their metastases. We report a case of a 28-yr-old woman with ectopic Cushing's syndrome due to an ACTH-secreting bronchial carcinoid, in whom the use of radioguided surgery improved disease management. At presentation, radiolabeled pentetreotide scintigraphy was the only procedure able to detect the ectopic source of ACTH. After radiologic confirmation, the patient underwent removal of a bronchial carcinoid, with disease persistence. After surgery, pentetreotide scintigraphy showed pathologic uptake in the mediastinum not previously detected at surgery and only subsequently confirmed by radiologic studies. Despite a second thoracic exploration, hormonal, scintigraphic, and radiological evidence of residual disease persisted. Radioguided surgery was then performed using a hand-held gamma probe 48 h after iv administration of a tracer dose of radiolabeled [111In-DTPA-D-Phe1]-pentetreotide, which permitted detection and removal of multiple residual mediastinal lymph node metastases. Clinical and radiologic cure, with no evidence of tracer uptake at pentetreotide scintigraphy, was subsequently observed. The use of an intraoperative gamma counter appears a promising procedure in the management of metastatic ACTH-secreting bronchial carcinoids. \ua92005, Editrice Kurtis