Isabella Castellano

Somatic ATP1A1, ATP2B3, and KCNJ5 Mutations in Aldosterone-Producing Adenomas

Aldosterone-producing adenomas (APAs) cause a sporadic form of primary aldosteronism and somatic mutations in the KCNJ5 gene, which encodes the G-protein–activated inward rectifier K+ channel 4, GIRK4, account for ≈40% of APAs. Additional somatic APA mutations were identified recently in 2 other genes, ATP1A1 and ATP2B3, encoding Na+/K+-ATPase 1 and Ca2+-ATPase 3, respectively, at a combined prevalence of 6.8%. We have screened 112 APAs for mutations in known hotspots for genetic alterations associated with primary aldosteronism. Somatic mutations in ATP1A1, ATP2B3, and KCNJ5 were present in 6.3%, 0.9%, and 39.3% of APAs, respectively, and included 2 novel mutations (Na+/K+-ATPase p.Gly99Arg and GIRK4 p.Trp126Arg). CYP11B2 gene expression was higher in APAs harboring ATP1A1 and ATP2B3 mutations compared with those without these or KCNJ5 mutations. Overexpression of Na+/K+-ATPase p.Gly99Arg and GIRK4 p.Trp126Arg in HAC15 adrenal cells resulted in upregulation of CYP11B2 gene expression and its transcriptional regulator NR4A2. Structural modeling of the Na+/K+-ATPase showed that the Gly99Arg substitution most likely interferes with the gateway to the ion binding pocket. In vitro functional assays demonstrated that Gly99Arg displays severely impaired ATPase activity, a reduced apparent affinity for Na+ activation of phosphorylation and K+ inhibition of phosphorylation that indicate decreased Na+ and K+ binding, respectively. Moreover, whole cell patch-clamp studies established that overexpression of Na+/K+-ATPase Gly99Arg causes membrane voltage depolarization. In conclusion, somatic mutations are common in APAs that result in an increase in CYP11B2 gene expression and may account for the dysregulated aldosterone production in a subset of patients with sporadic primary aldosteronism.

Variations in sentinel node isolated tumour cells/micrometastasis and non-sentinel node involvement rates according to different interpretations of the TNM definitions

Breast cancers with nodal isolated tumour cells (ITC) and micrometastases are categorised as node-negative and node-positive, respectively, in the tumour node metastasis (TNM) classification. Two recently published interpretations of the TNM definitions were applied to cases of low-volume sentinel lymph node (SLN) involvement and their corresponding non-SLNs for reclassification as micrometastasis or ITC. Of the 517 cases reviewed, 82 had ITC and 435 had micrometastasis on the basis of one classification, and the number of ITC increased to 207 with 310 micrometastases on the basis of the other. Approximately 24% of the cases were discordantly categorised. The rates of non-SLN metastases associated with SLN ITCs were 8.5% and 13.5%, respectively. Although the second interpretation of low-volume nodal stage categories has better reproducibility, it may underestimate the rate of non-SLN involvement. The TNM definitions of low-volume nodal metastases need to be better formulated and supplemented with visual information in the form of multiple sample images.

Routine assessment of prognostic factors in breast cancer using a multicore tissue microarray procedure

We propose multicore tissue microarray (TMA) as an alternative to whole section for routine assessment of prognostic factors in breast cancer. Since 2004, we introduced the multicore TMA for testing estrogen (ER) and progesterone receptors (PR), proliferation activity by Ki67, and HER2 overexpression and amplification in routine work. At least four tumor foci were selected on the whole section, and a dedicated technician used a stereomicroscope for accurate sampling of the selected areas. To identify a specific case in the TMA, a separate file and a computerized reporting form with the TMA map were created. A preliminary pilot study comparing the TMA results with those obtained on whole sections showed the specificity of the procedure. Moreover, in everyday diagnosis, hormone receptors were repeated on full section when negative in TMA, without significant discrepancy. Retrospective analysis of the 237 breast carcinomas studied by TMA showed the expected correspondence of tumor-grade differentiation with the hormone receptor pattern, the proliferation activity, and HER2 immunohistochemical and FISH values. In conclusion, multicore TMA may be an efficient approach in the routine study of prognostic factors in breast cancer, significantly reducing costs, time, and burden of slides necessary to accomplish these mandatory tests.

Estrogen receptor-β is expressed in stromal cells of fibroadenoma and phyllodes tumors of the breast

An estrogen dependency has been suggested for the growth of fibroadenomas: however, thus far, none of the steroid hormone receptors acting on breast tissues has been demonstrated in the stroma of breast fibroepithelial lesions. In this study, the expression of estrogen receptor (ER)-α and -β was investigated by immunohistochemistry in 33 fibroadenomas and in 30 benign, three borderline and seven malignant phyllodes tumors, all with spindle cell growth and in one distant metastasis. In addition, the presence of ER-β mRNA and its variants was evaluated by RT-PCR in microdissected stroma. The possible correlation between hormone receptor expression and differentiation processes of stromal cells was investigated by smooth muscle actin and calponin immunostaining. ER-β was the only hormone receptor expressed by stroma of fibroadenomas and phyllodes tumors, both at protein and mRNA level. The highest percentage of ER-β was observed in fibroadenomas with cellular stroma and in phyllodes tumors. In both lesions, ER-β-positive stromal cells showed expression of smooth muscle actin and/or calponin, as demonstrated by double immunostaining. In addition, the mean age at diagnosis was significantly lower in patients with ER-β-positive vs ER-β-negative fibroadenomas. In contrast, in phyllodes tumors, ER-β expression was higher in older patients. In conclusion, (i) only ER-β is detected in the stroma of fibroadenomas and phyllodes tumors; (ii) its expression correlates with the expression of smooth muscle markers and suggests a role of ER-β in myofibroblastic differentiation of stromal cells. These two results, together with the young age of patients carrying fibroadenomas with highly ER-β-positive stroma cells, may further indicate a hormone-receptor mechanism involved in regulating the growth of fibroadenomas. Conversely, the older age of patients with ER-β-rich phyllodes tumors suggests that mechanisms, probably independent from estrogen stimulation, act on the growth of these tumors.

Immunohistochemical, genetic and clinical characterization of sporadic aldosterone-producing adenomas.

Adrenal glands removed for unilateral primary aldosteronism (PA) display marked histological heterogeneity. Recently reported somatic mutations in KCNJ5, ATP1A1, ATP2B3 and CACNA1D can partially account for these differences. In this study we aimed at combining phenotypic and genotypic characteristics, integrating genetic and immunohistochemistry correlates in sporadic PA. Seventy-one adrenal glands have been included in the study and analyzed for mutations in KCNJ5, ATP1A1, ATP2B3 and CACNA1D. Histological examination and immunohistochemical staining for CYP11B1 (11β-hydroxylase) and CYP11B2 (aldosterone synthase) were performed on aldosterone-producing adenomas (APAs) and adjacent adrenal cortex. In our cohort, the final histopathological diagnosis was multinodular hyperplasia in 22.5% of the patients and single nodule in 77.5%. Forty-five percent of the removed adrenals displayed extra-APA CYP11B2-positive cell nests (B2-CN). Among adrenal vein sampling parameters the suppression of contralateral adrenal was more frequent and the lateralization index was higher in the subgroup of patients without extra-APA B2-CN compared to the subgroup with extra-APA B2-CN. KCNJ5-mutated APAs were composed mainly of zona fasciculata-like cells with high expression of CYP11B1, while ATP1A1, ATP2B3 and CACNA1D-mutated APAs presented more frequently a zona-glomerulosa-like phenotype with high expression of CYP11B2. We observed a significant inverse correlation between CYP11B2 expression and the size of the nodules and, if CYP11B2 expression was corrected for tumor volume, a significant correlation with plasma aldosterone and aldosterone to renin ratio. Our findings indicate that combination of genotyping and immunohistochemistry improves the final histopathological diagnosis between single nodule and multinodular hyperplasia of the assessed adrenals.

International Multicenter Tool to Predict the Risk of Nonsentinel Node Metastases in Breast Cancer

BACKGROUND Axillary treatment of breast cancer patients is undergoing a paradigm shift, as completion axillary lymph node dissections (ALNDs) are being questioned in the treatment of patients with tumor-positive sentinel nodes. This study aims to develop a novel multi-institutional predictive tool to calculate patient-specific risk of residual axillary disease after tumor-positive sentinel node biopsy. METHODS Breast cancer patients with a tumor-positive sentinel node and a completion ALND from five European centers formed the original patient series (N = 1000). Statistically significant variables predicting nonsentinel node involvement were identified in logistic regression analysis. A multivariable predictive model was developed and validated by area under the receiver operating characteristics curve (AUC), first internally in 500 additional patients and then externally in 1068 patients from other centers. All statistical tests were two-sided. RESULTS Nine tumor- and sentinel node-specific variables were identified as statistically significant factors predicting nonsentinel node involvement in logistic regression analysis. A resulting predictive model applied to the internal validation series resulted in an AUC of 0.714 (95% confidence interval [CI] = 0.665 to 0.763). For the external validation series, the AUC was 0.719 (95% CI = 0.689 to 0.750). The model was well calibrated in the external validation series. CONCLUSIONS We present a novel, international, multicenter, predictive tool to assess the risk of additional axillary metastases after tumor-positive sentinel node biopsy in breast cancer. The predictive model performed well in internal and external validation but needs to be further studied in each center before application to clinical use.

Reliability of whole sentinel lymph node analysis by one-step nucleic acid amplification for intraoperative diagnosis of breast cancer metastases.

Objective: To assess the reliability of using the One-Step Nucleic Acid Amplification (OSNA) assay as a single test on whole sentinel lymph nodes (SLN) as a method of intraoperative diagnosis and staging of SLNs in breast cancer. Background: Combining histological and molecular assessment of metastasis on the same SLN may not fully reproduce the actual load of cancer cells present in the SLN and create problems in decisions regarding axillary dissection. Methods: Selection criteria for the whole SLN OSNA test required that the primary tumor expressed CK19 in more than 80% of tumor cells. Imprint cytology analysis of SLNs was performed together with the OSNA. Results: Of the 279 patients enrolled for SLN evaluation, 123 gave consent to the OSNA protocol and 156 to the standard histology. Thirteen patients were excluded from OSNA evaluation because of low CK19 gene expression in the primary tumor; only 2.3% were truly negative. The kappa of concordance between the imprint cytology and OSNA results was 0.52. The rate of macrometastases determined by OSNA was 11% versus 20% determined by histology, whereas the rate of OSNA-micrometastases (18%) was significantly higher than that determined by histology (8%). The rate of SLN-negative cases was similar between the 2 protocols. Macrometastases correlated with the presence of vascular invasion in both protocols. The rate of axillary lymph node metastases was consistent with SLN tumor load. Conclusions: Intraoperative OSNA assay performed on the whole SLN gave objective and reproducible results that were useful for directing decisions regarding axillary dissection and for accurately defining the SLN stage.

Comparison of Positron Emission Tomography Scanning and Sentinel Node Biopsy in the Detection of Inguinal Node Metastases in Patients With Anal Cancer

BACKGROUND Inguinal lymph node metastases in patients with anal cancer are an independent prognostic factor for local failure and overall mortality. Inguinal lymph node status can be adequately assessed with sentinel node biopsy, and the radiotherapy strategy can subsequently be changed. We compared this technique vs. dedicated 18F-fluorodeoxyglucose positron emission tomography (PET) to determine which was the better tool for staging inguinal lymph nodes. METHODS AND MATERIALS In our department, 27 patients (9 men and 18 women) underwent both inguinal sentinel node biopsy and PET-CT. PET-CT was performed before treatment and then at 1 and 3 months after treatment. RESULTS PET-CT scans detected no inguinal metastases in 20 of 27 patients and metastases in the remaining 7. Histologic analysis of the sentinel lymph node detected metastases in only three patients (four PET-CT false positives). HIV status was not found to influence the results. None of the patients negative at sentinel node biopsy developed metastases during the follow-up period. PET-CT had a sensitivity of 100%, with a negative predictive value of 100%. Owing to the high number of false positives, PET-CT specificity was 83%, and positive predictive value was 43%. CONCLUSIONS In this series of patients with anal cancer, inguinal sentinel node biopsy was superior to PET-CT for staging inguinal lymph nodes.

Accuracy of tumour size assessment in the preoperative staging of breast cancer: comparison of digital mammography, tomosynthesis, ultrasound and MRI

PurposeAccurate measurement of breast tumour size is fundamental for treatment planning. We compared the accuracy of digital mammography (DM), digital breast tomosynthesis (DBT), ultrasound (US) and magnetic resonance imaging (MRI) for the preoperative evaluation of breast cancer size.Materials and methodsWe retrospectively reviewed 149 breast cancers in 110 patients who underwent DM, DBT, US and MRI between January 2010 and December 2011, before definitive surgery. The lesions were measured by two radiologists, without knowledge of the final histological examination, considered the gold standard. For each imaging modality, the maximum tumour size was measured to the nearest millimetre; the measurements were considered concordant if they were within ±5 mm. Pearson’s correlation coefficient was calculated for each imaging modality.ResultsThe median pathological tumour size was 22.3 mm. MRI and DBT had a level of concordance with pathology of 70% and 66%, respectively, which was higher than that of DM (54%). DBT and MRI measurements had a better correlation with pathological tumour size (R:0.89 and R:0.92, respectively) compared to DM (R:0.83) and US (R:0.77).ConclusionsDBT and MRI are superior to DM and US in the preoperative assessment of breast tumour size. DBT seems to improve the accuracy of DM, although MRI remains the most accurate imaging modality for breast cancer extension.RiassuntoObiettivoLa corretta valutazione pre-operatoria dell’estensione del carcinoma mammario è fondamentale nella pianificazione chirurgica-terapeutica. Scopo dello studio è stato confrontare l’accuratezza di mammografia digitale (DM), tomosintesi (DBT), ecografia (US) e risonanza magnetica (RM) nella valutazione preoperatoria delle dimensioni del tumore mammario rispetto all’istologia definitiva.Materiali e metodiSono stati valutati retrospettivamente 149 carcinomi in 110 pazienti, sottoposte a DM, DBT, US e RM prima dell’intervento chirurgico tra gennaio 2010 e dicembre 2011. Le lesioni sono state rivalutate e misurate da due radiologi non a conoscenza dell’estensione istologica. Le dimensioni valutate all’esame istologico sono state utilizzate come gold standard. Per ogni metodica, è stata valutata l’estensione massima tumorale; le misurazioni sono state considerate concordanti con l’istologia se comprese nei ±5 mm. è stato calcolato il coefficiente di correlazione di Pearson rispetto all’estensione istologica per ciascuna metodica.RisultatiIl diametro medio delle lesioni è stato 22,3 mm. RM e DBT hanno avuto rispettivamente una concordanza del 70% e 66% con l’istologia, superiore alla DM (54%). La DBT e l’RM hanno avuto un coefficiente di correlazione di Pearson con l’istologico definitivo(R: 0,89 e R: 0,92 rispettivamente) più elevato rispetto a DM (R: 0,83) e US (R: 0,77).ConclusioniDBT e RM sono risultate superiori a DM e US nella valutazione pre-operatoria dell’estensione tumorale. L’impiego della DBT sembra implementare i risultati della DM, sebbene l’RM rimanga la metodica di riferimento.

Distribution pattern of the Ki67 labelling index in breast cancer and its implications for choosing cut-off values

The Ki67 labelling index (LI - proportion of staining cells) is widely used to reflect proliferation in breast carcinomas. Several cut-off values have been suggested to distinguish between tumours with low and high proliferative activity. The aim of the current study was to evaluate the distribution of Ki67 LIs in breast carcinomas diagnosed at different institutions by different pathologists using the method reflecting their daily practice. Pathologists using Ki67 were asked to provide data (including the LI, type of the specimen, receptor status, grade) on 100 consecutively stained cases, as well as details of their evaluation. A full dataset of 1709 carcinomas was collected from 19 departments. The median Ki67 LI was 17% for all tumours and 14% for oestrogen receptor-positive and HER2-negative carcinomas. Tumours with higher mitotic counts were associated with higher Ki67 LIs. Ki67 LIs tended to cluster around values ending with 5 or 0 both in cases where the values were obtained by counting the proportion of stained tumour cell nuclei and those where the values were obtained by estimation. On the basis of the distribution pattern described, some currently used Ki67 LI cut off values are not realistic, and it is proposed to select more realistic values ending with 0 or 5.