Eleonora Duregon

Diagnostic and prognostic role of steroidogenic factor 1 in adrenocortical carcinoma: a validation study focusing on clinical and pathologic correlates

The pathologic characterization of adrenocortical cancer is still problematic for several reasons, including the identification of novel markers of diagnostic or prognostic relevance. Among them, steroidogenic factor 1 deserves major interest because of its potential usefulness as a marker of adrenocortical derivation and of biological aggressiveness. Our aim was to validate its prognostic relevance in a large series of adrenocortical cancer, comparing the performance of 2 different commercial antibodies and investigating its expression in adrenocortical cancer variants and in comparison with clinical and pathologic features. Seventy-five (including 53 classical, 10 myxoid, and 12 oncocytic) adrenocortical cancer cases were included in tissue microarrays and analyzed for the immunohistochemical expression of steroidogenic factor 1 using 2 commercial antibodies, 1 polyclonal and 1 monoclonal (N1665). Nuclear steroidogenic factor 1 staining was assessed using a semiquantitative score and correlated with adrenocortical cancer type and clinical pathologic characteristics. A weak but significant correlation was found comparing the 2 antibodies with a positive rate of 88% and 58% using the monoclonal and polyclonal antibodies, respectively. High steroidogenic factor 1 expression with the N1665 antibody was positively correlated with high mitotic count, high Ki-67 index, and high European Network for the Study of Adrenal Tumors (ENSAT) stage and negatively associated with loss of functionality and presence of oncocytic features. Moreover, high steroidogenic factor 1 expression with this same antibody was significantly associated at univariate analysis with a decreased survival, together with high Ki-67 and mitotic indexes, with a trend to significance confirmed by multivariate analysis, thus supporting the detection of steroidogenic factor 1 using the N1665 antibody as a novel prognostic marker in adrenocortical cancer.

Adrenocortical tumors with myxoid features: a distinct morphologic and phenotypical variant exhibiting malignant behavior.

Myxoid changes have been rarely reported both in adrenocortical adenomas and carcinomas. The recent observation by our group of an adrenal myxoid tumor with morphologically borderline features, but aggressive clinical behavior prompted us to review a series of 196 adrenocortical lesions, comprising 122 carcinomas and 74 adenomas, to define the morphologic, phenotypical and clinical characteristics of adrenocortical tumors with myxoid features. Fourteen cases, including 12 carcinomas and 2 borderline tumors, formed the basis of this report, and were characterized by a variably abundant myxoid component (from 5% to 90% of tumor) and 2 distinct cellular growth patterns: the first (10 cases), mostly associated with a predominant myxoid stromal component, was made of small cells with mild atypia arranged in cords and microcysts; the second (4 cases) was characterized by focal myxoid changes in tumors otherwise similar to conventional adrenocortical carcinoma, with large atypical cells having an eosinophilic cytoplasm and a diffuse or nodular architecture. The above mentioned patterns were absent in all adenomas reviewed. A peculiar reactivity to neurofilaments was seen, mostly associated to the presence of predominant rather that focal myxoid stromal changes, and in 40% of conventional adrenocortical carcinomas, thus representing an undescribed potential pitfall in the differential diagnosis of adrenal lesions. Myxoid adrenocortical tumors probably represent a rare but histologically and phenotipically distinct entity and, although rare cases of benign lesions are on record, they seem to be generally associated to morphologic and clinical features of malignancy.

ELOVL5 Mutations Cause Spinocerebellar Ataxia 38

Spinocerebellar ataxias (SCAs) are a heterogeneous group of autosomal-dominant neurodegenerative disorders involving the cerebellum and 23 different genes. We mapped SCA38 to a 56 Mb region on chromosome 6p in a SCA-affected Italian family by whole-genome linkage analysis. Targeted resequencing identified a single missense mutation (c.689G>T [p.Gly230Val]) in ELOVL5. Mutation screening of 456 independent SCA-affected individuals identified the same mutation in two further unrelated Italian families. Haplotyping showed that at least two of the three families shared a common ancestor. One further missense variant (c.214C>G [p.Leu72Val]) was found in a French family. Both missense changes affect conserved amino acids, are predicted to be damaging by multiple bioinformatics tools, and were not identified in ethnically matched controls or within variant databases. ELOVL5 encodes an elongase involved in the synthesis of polyunsaturated fatty acids of the ω3 and ω6 series. Arachidonic acid and docosahexaenoic acid, two final products of the enzyme, were reduced in the serum of affected individuals. Immunohistochemistry on control mice and human brain demonstrated high levels in Purkinje cells. In transfection experiments, subcellular localization of altered ELOVL5 showed a perinuclear distribution with a signal increase in the Golgi compartment, whereas the wild-type showed a widespread signal in the endoplasmic reticulum. SCA38 and SCA34 are examples of SCAs due to mutations in elongase-encoding genes, emphasizing the importance of fatty-acid metabolism in neurological diseases.

The Reticulin Algorithm for Adrenocortical Tumor Diagnosis: A Multicentric Validation Study on 245 Unpublished Cases.

The pathologic diagnosis of adrenocortical carcinoma (ACC) still needs to be improved, because the renowned Weiss Score (WS) system has a poor reproducibility of some parameters and is difficult to apply in borderline cases and in ACC variants. The “reticulin algorithm” (RA) defines malignancy through an altered reticulin framework associated with 1 of the 3 following parameter: necrosis, high mitotic rate, and vascular invasion. This study aimed at validating the interobserver reproducibility of reticulin stain evaluation in an unpublished series of 245 adrenocortical tumors (61 adenomas and 184 carcinomas) from 5 Italian centers, classified according to the WS. Eight pathologists reviewed all reticulin-stained slides. After training, a second round of evaluation on discordant cases was performed 10 weeks later. The RA reclassified 67 cases (27%) as adenomas, including 44 with no reticulin alterations and 23 with an altered reticulin framework but lacking the subsequent parameters of the triad. The other 178 cases (73%) were carcinomas according to the above-mentioned criteria. A complete (8/8 pathologists) interobserver agreement was reached in 75% of cases (&kgr;=0.702), irrespective of case derivation, pathologists’ experience, and histologic variants, and was further improved when only those cases with high WS and clinically malignant behavior were considered. After the training, the overall agreement increased to 86%. We conclude that reticulin staining is a reliable technique and an easy-to-interpret system in adrenocortical tumors; moreover, it has a high interobserver reproducibility, which supports the notion of using such a method in the proposed 2-step RA approach for ACC diagnosis.

YKL-40/c-Met expression in rectal cancer biopsies predicts tumor regression following neoadjuvant chemoradiotherapy: a multi-institutional study.

Background Neoadjuvant chemo-radiotherapy (CRT) followed by surgical resection is the standard treatment for locally advanced rectal cancer, although complete tumor pathological regression is achieved in only up to 30% of cases. A clinicopathological and molecular predictive stratification of patients with advanced rectal cancer is still lacking. Here, c-Met and YKL-40 have been studied as putative predictors of CRT response in rectal cancer, due to their reported involvement in chemoradioresistance in various solid tumors. Material and Methods A multicentric study was designed to assess the role of c-Met and YKL-40 expression in predicting chemoradioresistance and to correlate clinical and pathological features with CRT response. Immunohistochemistry and fluorescent in situ hybridization for c-Met were performed on 81 rectal cancer biopsies from patients with locally advanced rectal adenocarcinoma. All patients underwent standard (50.4 gy in 28 fractions + concurrent capecitabine 825 mg/m2) neoadjuvant CRT or the XELOXART protocol. CRT response was documented on surgical resection specimens and recorded as tumor regression grade (TRG) according to the Mandard criteria. Results A significant correlation between c-Met and YKL-40 expression was observed (R = 0.43). The expressions of c-Met and YKL-40 were both significantly associated with a lack of complete response (86% and 87% of c-Met and YKL-40 positive cases, p< 0.01 and p = 0.006, respectively). Thirty of the 32 biopsies co-expressing both markers had partial or absent tumor response (TRG 2-5), strengthening their positive predictive value (94%). The exclusive predictive role of YKL-40 and c-Met was confirmed using a multivariate analysis (p = 0.004 and p = 0.007 for YKL-40 and c-Met, respectively). TRG was the sole morphological parameter associated with poor outcome. Conclusion c-Met and YKL-40 expression is a reliable predictor of partial/absent response to neoadjuvant CRT in rectal cancer. Targeted therapy protocols could take advantage of prior evaluations of c-MET and YKL-40 expression levels to increase therapeutic efficacy.

Comparative diagnostic and prognostic performances of the hematoxylin-eosin and phospho-histone H3 mitotic count and Ki-67 index in adrenocortical carcinoma

Mitotic count on hematoxylin and eosin slides is a fundamental morphological criterion in the diagnosis and grading of adrenocortical carcinoma in any scoring system employed. Moreover, it is the unique term strongly associated with patient’s prognosis. Phospho-histone H3 is a mitosis-specific antibody, which was already proven to facilitate mitotic count in melanoma and other tumors. Therefore, a study was designed to assess the diagnostic and prognostic role of phospho-histone H3 in 52 adrenocortical carcinomas, comparing manual and computerized count to standard manual hematoxylin- and eosin-based method and Ki-67 index. Manual hematoxylin and eosin and phospho-histone H3 mitotic counts were highly correlated (r=0.9077, P<0.0001), better than computer-assisted phospho-histone H3 evaluations, and had an excellent inter-observer reproducibility at Bland-Altman analysis. Three of 15 cases having <5 mitotic figures per 50 high-power fields by standard count on hematoxylin and eosin gained the mitotic figure point of Weiss Score after a manual count on phospho-histone H3 slides. Traditional mitotic count confirmed to be a strong predictor of overall survival (P=0.0043), better than phospho-histone H3-based evaluation (P=0.051), but not as strong as the Ki-67 index (P<0.0001). The latter further segregated adrenocortical carcinomas into three prognostic groups, stratifying cases by low (<20%), intermediate (20–50%), and high (>50%) Ki-67 values. We conclude that (a) phospho-histone H3 staining is a useful diagnostic complementary tool to standard hematoxylin and eosin mitotic count, enabling optimal mitotic figure evaluation (including atypical mitotic figures) even in adrenocortical carcinomas with a low mitotic index and with a very high reproducibility; (b) Ki-67 proved to be the best prognostic indicator of overall survival, being superior to the mitotic index, irrespective of the method (standard on hematoxylin and eosin or phospho-histone H3-based) used to count mitotic figures.

Oncocytic Adrenocortical Tumors: Diagnostic Algorithm and Mitochondrial DNA Profile in 27 Cases.

The pathologic diagnosis of adrenocortical carcinoma (ACC) relies on microscopic features that are sometimes equivocal in special variants, including oncocytic adrenocortical tumors (OACTs). We report a series of 27 unpublished OACTs (15 pure and 12 mixed or focal) and assess for the first time in OACTs the diagnostic utility of an algorithm recently proposed by our group (“reticulin” algorithm) for conventional ACCs on the basis of a combination of reticulin staining and assessment of only 3 Weiss parameters. Overall, 12 cases were malignant according to the Lin-Weiss-Bisceglia (L-W-B) system for pure tumors and the original Weiss system for mixed or focal tumors; extensive or focal disruption of the reticulin network was found in 16 of 27 OACTs. This was associated with either a high mitotic index, presence of necrosis, and/or vascular invasion in 14 of these, which were thus considered malignant according to our algorithm. From a clinical standpoint, OACTs, at least in the pure form, are “low grade” lesions with a low mean Weiss score, mitotic and Ki-67 indices, and uncommon capsular or vascular invasion. They, including unequivocal morphologically malignant cases, generally pursue an indolent clinical course. In addition, the 4977 bp mitochondrial DNA “common deletion” was detected using real-time polymerase chain reaction in 54% of cases from this study and an additional validation series of 23 OACTs, with a heterogenous (heteroplasmic) intratissue and intracellular distribution (as detected by a modified FISH procedure) and a marked association with the presence of intact reticulin framework.

microRNA expression patterns in adrenocortical carcinoma variants and clinical pathologic correlations

Several microRNAs (miRNAs) were shown to be deregulated in adrenocortical carcinoma (ACC) as compared with adenoma, but a detailed assessment of their expression in its histologic variants and correlation with clinicopathologic characteristics has not been performed, so far. Our aim was to assess the expression of 5 selected miRNAs (IGF2 gene-related miR-483-3p and 5p and hypoxia-induced miR-210, miR-195, and miR-1974) in a series of 51 ACCs (35 classical, 6 myxoid, and 10 oncocytic) as compared with clinical and pathologic features and immunohistochemical expression of prognostic markers, including steroidogenic factor 1, p53, β-catenin, and glucose transporter 1. Oncocytic carcinomas had a reduced expression of miR-483-3p (P = .0325), miR-483-5p (P = .0175), and miR-210 (P = .0366), as compared with other histotypes. Overexpression of miR-210 was associated with the presence of necrosis (P = .0035), high Ki-67 index (P = .0013), and high glucose transporter 1 expression (P = .0043), whereas an inverse correlation with mitotic rate was observed in cases with high miR-493-3p (P = .0191) and miR-1974 (P = .0017) expression. High miR-1974 was also associated with low Ki-67 (P = .0312) and European Network for the Study of Adrenal Tumors stage (P = .0082) and negative p53 (P = .0013). At univariate analysis myxoid/classic histotype (P = .026), high miR-210 (P = .0465), high steroidogenic factor 1 protein (P = .0017), high Ki-67 (P = .0066), and high mitotic index (P = .0006) were significantly associated the shorter overall survival, the latter being the sole independent prognostic factor at multivariate analysis (P = .017). In conclusion, (a) miR-483-3p, miR-483-5p, and miR-210 are differentially expressed in ACC variants, and (b) high miR-210 is associated with clinicopathologic parameters of aggressiveness and a poor prognosis.

An International Ki67 Reproducibility Study in Adrenal Cortical Carcinoma.

Despite the established role of Ki67 labeling index in prognostic stratification of adrenocortical carcinomas and its recent integration into treatment flow charts, the reproducibility of the assessment method has not been determined. The aim of this study was to investigate interobserver variability among endocrine pathologists using a web-based virtual microscopy approach. Ki67-stained slides of 76 adrenocortical carcinomas were analyzed independently by 14 observers, each according to their method of preference including eyeballing, formal manual counting, and digital image analysis. The interobserver variation was statistically significant (P<0.001) in the absence of any correlation between the various methods. Subsequently, 61 static images were distributed among 15 observers who were instructed to follow a category-based scoring approach. Low levels of interobserver (F=6.99; Fcrit=1.70; P<0.001) as well as intraobserver concordance (n=11; Cohen &kgr; ranging from −0.057 to 0.361) were detected. To improve harmonization of Ki67 analysis, we tested the utility of an open-source Galaxy virtual machine application, namely Automated Selection of Hotspots, in 61 virtual slides. The software-provided Ki67 values were validated by digital image analysis in identical images, displaying a strong correlation of 0.96 (P<0.0001) and dividing the cases into 3 classes (cutoffs of 0%-15%-30% and/or 0%-10%-20%) with significantly different overall survivals (P<0.05). We conclude that current practices in Ki67 scoring assessment vary greatly, and interobserver variation sets particular limitations to its clinical utility, especially around clinically relevant cutoff values. Novel digital microscopy–enabled methods could provide critical aid in reducing variation, increasing reproducibility, and improving reliability in the clinical setting.

BRCA1-Associated Protein 1 (BAP1) Immunohistochemical Expression as a Diagnostic Tool in Malignant Pleural Mesothelioma Classification: A Large Retrospective Study

Introduction Malignant pleural mesothelioma (MPM) is a highly aggressive disease with limited therapeutic options. Histological subtype remains among the most reliable prognostic factors, because the epithelioid subtype associated with the best prognosis and the sarcomatoid subtype with the worst. The biphasic subtype has an intermediate prognosis, but its definitive histological diagnosis may be challenging owing to the difficulty of assessing the neoplastic nature of the stromal component. Recent data identified BRCA1‐associated protein 1 gene (BAP1) as one of the most frequently mutated genes in MPM. Immunohistochemical testing for BRCA1‐associated protein 1 (BAP1) has been proposed to be predictive for the detection of BAP1 mutation in neoplastic cells. The aim of the present study was to define the diagnostic usefulness of immunohistochemical determination of BAP1 in MPM, with clinicopathological correlation. Methods A series of 143 MPMs were investigated for BAP1 protein expression in correlation with clinical and pathological data, including with a newly proposed nuclear grade. A pilot series of 20 selected cases were also investigated for BAP1 mutational status. Results Negative nuclear staining for BAP1 occurred in 62% of MPMs (including 27% with a cytoplasmic pattern) and was significantly associated with the presence of BAP1 mutation, epithelioid subtype, and a better prognosis. In a subgroup of cases, the pattern of expression of BAP1 in stromal cells supported their distinction as reactive versus neoplastic, thus helping achieve the correct classification of biphasic histological subtype. Conclusions We showed that BAP1 protein determination is a diagnostic tool to correctly distinguish biphasic MPM from epithelial subtypes with an atypical/activated reactive stroma and an independent prognostic parameter in MPM.