Chiara Cantaloni

Proliferative activity in human breast cancer: Ki-67 automated evaluation and the influence of different Ki-67 equivalent antibodies.

BackgroundKi67 labeling index (Ki67 LI), the percentage Ki67 immunoreactive cells, is a measure of tumor proliferation, with important clinical relevance in breast cancer, and it is extremely important to standardize its evaluation.AimTo test the efficacy of computer assisted image analysis (CAIA) applied to completely digitized slides and to assess its feasibility in routine practice and compare the results obtained using two different Ki67 monoclonal antibodies.Materials and methods315 consecutive breast cancer routinely immunostained for Ki-67 (223 with SP6 and 92 with MM1 antibodies previously examined by an experienced pathologist, have been re-evaluated using Aperio Scanscope Xs.ResultsMean human Ki67 LI values were 36%± 14.% and 28% ± 18% respectively for SP6 and MM1 antibodies; mean CAM Ki67 LI values were 31%± 19% and 22% ± 18% respectively for SP6 and MM1. Human and CAIA evaluation are statistically highly correlated (Pearson: 0.859, p<0.0001), although human LI are systematically higher. An interobserver variation study on CAIA performed on 84 cases showed that the correlation between the two evaluations was linear to an excellent degree.DiscussionOur study shows that a) CAIA can be easily adopted in routine practice, b) human and CAIA Ki67 LI are highly correlated, although human LI are systematically higher, c) Ki67 LI using different evaluation methods and different antibodies shows important differences in cut-off values.

Molecular and clinicopathologic characterization of gastrointestinal stromal tumors (GISTs) of small size.

Although Gastrointestinal stromal tumors (GISTs) affect about 0.0014% of the population, GISTs smaller than 1 cm (microGISTs) are detectable in about 20% to 30% of elderly individuals. This suggests that microGISTs likely represent premalignant precursors that evolve only in a minute fraction of cases toward overt GISTs. We sought histopathologic and molecular explanations for the infrequent clinical progression in small GISTs. To investigate the mechanisms of GIST progression and identify subsets with differential malignant potential, we carried out a thorough characterization of 170 GISTs <2 cm and compared their KIT/PDGFRA status with overt GISTs. The proliferation was lower in microGISTs compared with GISTs from 1 to 2 cm (milliGISTs). In addition, microGISTs were more frequently incidental, gastric, spindle, showed an infiltrative growth pattern, a lower degree of cellularity, and abundant sclerosis. The progression was limited to 1 ileal and 1 rectal milliGISTs. KIT/PDGFRA mutations were detected in 74% of the cases. The overall frequency of KIT/PDGFRA mutation and, particularly, the frequency of KIT exon 11 mutations was significantly lower in small GISTs compared with overt GISTs. Five novel mutations, 3 in KIT (p.Phe506Leu, p.Ser692Leu, p.Glu695Lys) 2 in PDGFRA (p.Ser847X, p.Ser667Pro), plus 4 double mutations were identified. Small GISTs share with overt GIST KIT/PDGFRA mutation. Nevertheless, microGISTs display an overall lower frequency of mutations, particularly canonical KIT mutations, and also carry rare and novel mutations. These molecular features, together with the peculiar pathologic characteristics, suggest that the proliferation of these lesions is likely sustained by weakly pathogenic molecular events, supporting the epidemiologic evidence that microGISTs are self-limiting lesions.

miR-205 Expression Levels in Nonsmall Cell Lung Cancer Do Not Always Distinguish Adenocarcinomas From Squamous Cell Carcinomas.

Accurate classification of nonsmall cell lung cancers is of paramount clinical relevance, as novel chemotherapeutic agents show different efficacy in adenocarcinomas (ADCs) compared with squamous cell carcinomas (SQCCs). Cyto and histomorphology may sometimes be insufficient for this distinction and immunohistochemistry may improve diagnostic accuracy. The measurement of miR-205 may be another tool for the distinction between ADC and SQCC. The aim of our study was to compare morphologic and immunohistochemical classification with the relative quantification of miR-205 and miR-21 insurgically resected and well-characterized lung tumors (25 ADCs, 24 SQCCs, 1 adenosquamous). The miR-21 relative levels were similar in SQCC and ADC, whereas the miR-205 relative levels were lower in ADC (P<0.0001). The miR-205 sample score value, determined according to Lebanony et al, was higher in ADC (range, 2.8 to 9.08) compared with SQCC (range, −4.17 to 2.445) (P<0.0001). Accordingly, 22 tumors were classified as ADC and 28 tumors as SQCC, although 8 cases (2 SQCCs and 6 ADCs) were in the range of “near cutoff values.” Four cases classified as SQCC (according to the sample score method) corresponded to cases classified as ADC on the basis of morphoimmunohistochemical evaluation. In conclusion, the relative quantification of miR-205 and miR-21 seems to be a promising diagnostic tool. However, the molecular approach is still not completely satisfactory as it may misclassify a non-negligible percentage of cases. Therefore, it cannot be used as a substitute of accurate morphologic and immunophenotypical characterization of tumors, but could be used as an adjunctive diagnostic criterion in selected cases.

Heterogeneity of large cell carcinoma of the lung: an immunophenotypic and miRNA-based analysis.

Large cell carcinomas (LCCs) of the lung are heterogeneous and may be of different cell lineages. We analyzed 56 surgically resected lung tumors classified as LCC on the basis of pure morphologic grounds, using a panel of immunophenotypic markers (adenocarcinoma [ADC]-specific, thyroid transcription factor-1, cytokeratin 7, and napsin A; squamous cell carcinoma [SQCC]-specific, p63, cytokeratin 5, desmocollin 3, and Δnp63) and the quantitative analysis of microRNA-205 (microRNA sample score [mRSS]). Based on immunoprofiles 19 (34%) of the cases were reclassified as ADC and 14 (25%) as SQCC; 23 (41%) of the cases were unclassifiable. Of these 23 cases, 18 were classified as ADC and 5 as SQCC according to the mRSS. Our data show that an extended panel of immunohistochemical markers can reclassify around 60% of LCCs as ADC or SQCC. However, a relevant percentage of LCCs may escape convincing immunohistochemical classification, and mRSS could be used for further typing, but its clinical relevance needs further confirmation.

Recurrent Prostate Cancer Genomic Alterations Predict Response to Brachytherapy Treatment

Background: This study aimed to evaluate the association of recurrent molecular alterations in prostate cancer, such as ERG rearrangements and phosphatase and tensin homolog gene (PTEN) deletions, with oncologic outcomes in patients with prostate cancer treated with brachytherapy. Methods: Ninety-two men underwent I-125 brachytherapy with a 145 Gy delivered dose between 2000 and 2008. Pretreatment prostate biopsies were analyzed by immunohistochemistry (IHC) and FISH for ERG rearrangement and overexpression, PTEN deletion, and expression loss. Univariable and multivariable Cox-regression analyses evaluated association of ERG and PTEN status with biochemical recurrence (BCR). Results: Within a median follow-up of 73 months, 11% of patients experienced BCR. Of 80 samples with both IHC and FISH performed for ERG, 46 (57.8%) demonstrated rearrangement by FISH and 45 (56.3%) by IHC. Of 77 samples with both IHC and FISH for PTEN, 14 (18.2%) had PTEN deletion by FISH and 22 (28.6%) by IHC. No significant associations were found between ERG, PTEN status, and clinicopathologic features. Patients with concurrent ERG rearrangement and PTEN deletion demonstrated significantly worse relapse-free survival rates compared with those with ERG or PTEN wild type (P < 0.01). In multivariable Cox regression analysis adjusted for the effects of standard clinicopathologic features, combined ERG rearranged and PTEN deletion was independently associated with BCR (HR = 2.6; P = 0.02). Conclusions: Concurrent ERG rearrangement and PTEN loss was independently associated with time to BCR in patients undergoing brachytherapy. Future studies are needed to validate prostate cancer molecular subtyping for risk stratification. Impact: Identifying patients in the ERG-rearranged/PTEN-deleted molecular subclass may improve treatment personalization. Cancer Epidemiol Biomarkers Prev; 23(4); 594–600. ©2014 AACR.

Diagnostic value of automated Her2 evaluation in breast cancer: a study on 272 equivocal (score 2+) Her2 immunoreactive cases using an FDA approved system.

Goal of this study was to asses the performance of Aperio computer-assisted analysis for HER2 immunohistochemical measurement in 292 equivocally (score2+) HercepTest immunoreactive breast cancer cases, evaluated by an experienced pathologist and analyzed with fluorescent in situ hybridization (FISH). The automatic Aperio categorization and the percentage of immunoreactive cells as evaluated by the computer and by the pathologist were recorded. Computer-assisted analysis classified 7 (2.4%) cases as negative (0), 136 (46.6%) as faintly positive (1+), 134 (40.5%) as moderately positive (2+), and 15 (5.1%) as strongly positive (3+). Correlative component analysis (CCA) classification is associated with Her2 amplification (P<0.0001). Compared with the human evaluation, automated CCA classification would save 157 (58%) FISH analyses, while not identifying 15 amplified cases (6% false-negative rate). The mean computer percentage value (CPV) is 18.44% standard deviation ±19.00 (range, 0.01 to 76.10). CPV and the pathologist percentage value are significantly associated and correlated (P<0.001) and have similar sensitivity and specificity in identifying Her2 FISH-amplified cases. CPV has a very low interobserver variation. The difference in CPV in amplified and nonamplified subgroups is statistically significant (P<0.001). Receiver operating characteristic analysis indicates that CPV is good at separating FISH nonamplified from amplified cases (P<0.001). The optimal cut-off value maximizing both sensitivity and specificity is 17.6% (sensitivity=73.3%, specificity=71.6%). Using a different cut-off value (2% of positive cells) we would have missed only 3 amplified cases (1% false-negative rate) while not submitting to FISH 52 cases (18% of the whole series). This false-negative rate is well below the expected false-negative rate usually observed in score 1 cases, supporting the use of CCA with a modified cut-off value in routine diagnostics for equivocally stained HER2 cases.

Increased frequency of minimal homozygous deletions is associated with poor prognosis in primary malignant melanoma patients.

Identification of prognostic melanoma‐associated copy number alterations (CNAs) is still an area of active research. Here, we investigated by high‐resolution array comparative genomic hybridization (aCGH) a cohort of 31 paraffin‐preserved primary malignant melanomas (MMs), whose prognosis was not predictable on the basis of conventional histopathological parameters. Although we identified a variety of highly recurrent sites of genomic lesions, the total number of CNAs per patient was not a discriminator of MM outcome. Furthermore, validation of aCGH by quantitative PCR on an extended population of 65 MM samples confirmed the absence of predictive value for the most recurrent CNA loci. Instead, our analysis revealed specific prognostic potential of the frequency of homozygous deletions (representing less than 3% of the total CNAs on average per sample), which was strongly associated with sentinel lymph node (SLN) invasion (P = 0.003), and distant metastasis (P = 0.003). Increased number of homozygous deletions was also indicative of poor patient survival (P = 0.01), both in our samples and in an independent validation of public dataset of primary and metastatic MMs. Moreover, we identified 77 hotspots of minimal common homozygous deletions, enriched in genes involved in cell adhesion processes and cell‐communication functions, which preferentially accumulated in primary MMs showing the most severe outcome. Therefore, specific loss of gene loci in regions of minimal homozygous deletion may represent a pivotal type of genomic alteration accumulating during MM progression with potential prognostic implication.

Methods For Toxicity Testing Of Xenobiotics In Wastewater Treatment Plants And In Receiving Water Bodies

This paper focuses on conventional and advanced promising methods for the evaluation of toxicity originated by xenobiotics in WWTPs and surface waters. A concise review about toxicity tests applied to activated sludge and to receiving water bodies was presented. Advantages or limitations of the methods were discussed. Both heterotrophic and nitrifying activity were considered. Experimental results on the application to activated sludge of conventional toxicity tests (based on respirometry and bioluminescence) and advanced methods (based on the direct quantification of viable or dead bacteria by using flow cytometry) were presented in this paper. The advantage of respirometry and flow cytometry is related to the use of bacteria present in WWTPs, without need of using pure bacterial strains different from activated sludge.

Statistical analysis of a Bayesian classifier based on the expression of miRNAs

BackgroundDuring the last decade, many scientific works have concerned the possible use of miRNA levels as diagnostic and prognostic tools for different kinds of cancer. The development of reliable classifiers requires tackling several crucial aspects, some of which have been widely overlooked in the scientific literature: the distribution of the measured miRNA expressions and the statistical uncertainty that affects the parameters that characterize a classifier. In this paper, these topics are analysed in detail by discussing a model problem, i.e. the development of a Bayesian classifier that, on the basis of the expression of miR-205, miR-21 and snRNA U6, discriminates samples into two classes of pulmonary tumors: adenocarcinomas and squamous cell carcinomas.ResultsWe proved that the variance of miRNA expression triplicates is well described by a normal distribution and that triplicate averages also follow normal distributions. We provide a method to enhance a classifiers’ performance by exploiting the correlations between the class-discriminating miRNA and the expression of an additional normalized miRNA.ConclusionsBy exploiting the normal behavior of triplicate variances and averages, invalid samples (outliers) can be identified by checking their variability via chi-square test or their displacement by the respective population mean via Student’s t-test. Finally, the normal behavior allows to optimally set the Bayesian classifier and to determine its performance and the related uncertainty.

Unusual case of locally advanced and metastatic paratesticular liposarcoma: a case report and review of the literature

Liposarcoma accounts for 20% of all sarcomas and is a rare occurrence in the paratesticular region. We present the case of a 66-year-old man with a massive liposarcoma of the right scrotum invading the lower limb and the abdominal wall skin. The case concerns an unusually large and aggressive liposarcoma (25 cm), presenting with multiple lung and nodal metastases. The patient had an unfavourable evolution with rapid progression of metastases, although there were no signs of local disease. In this case, a wide local excision was performed in order to obtain local control of the disease. Even though paratesticular sarcomas might have a more favourable evolution, the association with lung involvement carries an ominous prognosis. Diagnosis of paratesticular sarcoma should be kept in mind in case of irregular necrotic masses in the inguinal and scrotal region.