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STEFANIA RAFFAELLA BETTELLI

Docente a contratto
Dipartimento Chirurgico, Medico, Odontoiatrico e di Scienze Morfologiche con interesse Trapiantologico, Oncologico e di Medicina Rigenerativa

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Pubblicazioni

2024 - Proteomics Analysis of Formalin-Fixed Paraffine-Embedded Tissue Reveals Key Proteins Related to Lung Dysfunction in Idiopathic Pulmonary Fibrosis. [Articolo su rivista]
Samarelli, ANNA VALERIA; Tonelli, Roberto; Raineri, Giulia; Bruzzi, Giulia; Andrisani, Dario; Gozzi, Filippo; Marchioni, Alessandro; Costantini, Matteo; Fabbiani, Luca; Genovese, Filippo; Pinetti, Diego; Manicardi, Linda; Castaniere, Ivana; Masciale, Valentina; Aramini, Beatrice; Tabbi', Luca; Rizzato, Simone; Bettelli, Stefania; Manfredini, Samantha; Dominici, Massimo; Clini, Enrico; Cerri, Stefania
abstract

Idiopathic pulmonary fibrosis (IPF) severely affects the lung leading to aberrant deposition of extracellular matrix and parenchymal stiffness with progressive functional derangement. The limited availability of fresh tissues represents one of the major limitations to study the molecular profiling of IPF lung tissue. The primary aim of this study was to explore the proteomic profiling yield of archived formalin-fixed paraffin-embedded (FFPE) specimens of IPF lung tissues. We further determined the protein expression according to respiratory functional decline at the time of biopsy. The total proteins isolated from 11 FFPE samples of IPF patients compared to 3 FFPE samples from a non-fibrotic lung defined as controls, were subjected to label-free quantitative proteomic analysis by liquid chromatography-mass spectrometry (LC-MS/MS) and resulted in the detection of about 400 proteins. After the pairwise comparison between controls and IPF, functional enrichment analysis identified differentially expressed proteins that were involved in extracellular matrix signaling pathways, focal adhesion and transforming growth factor β (TGF‐β) signaling pathways strongly associated with IPF onset and progression. Five proteins were significantly over-expressed in the lung of IPF patients with either advanced disease stage (Stage II) or impaired pulmonary function (FVC<75, DLco<55) compared to controls; these were lymphocyte cytosolic protein 1 (LCP1), peroxiredoxin-2 (PRDX2), transgelin 2 (TAGLN2), lumican (LUM) and mimecan (OGN) that might play a key role in the fibrogenic processes. Our work showed that the analysis of FFPE samples was able to identify key proteins that might be crucial for the IPF pathogenesis. These proteins are correlated with lung carcinogenesis or involved in the immune landscape of lung cancer, thus making possible common mechanisms between lung carcinogenesis and fibrosis progression, two pathological conditions at risk for each other in the real life.

2024 - Real-World Data and Clinical Implications of Next-Generation Sequencing (NGS)-Based Analysis in Metastatic Breast Cancer Patients [Articolo su rivista]
Canino, F; Tornincasa, A; Bettelli, S; Manfredini, S; Barbolini, M; Moscetti, L; Omarini, C; Toss, A; Tamburrano, F; Antonelli, G; Baglio, F; Belluzzi, L; Martinelli, G; Natalizio, S; Ponzoni, O; Dominici, M; Piacentini, F.
abstract

Over the last two decades, the use of Next-Generation Sequencing (NGS) in medical oncology has increased the likelihood of identifying druggable mutations that may be potentially susceptible to targeted treatments. The European Society for Medical Oncology (ESMO) currently does not recommend the use of the NGS test to determine the therapeutic course of patients with metastatic breast cancer (mBC) in daily clinical practice. However, the aim of this work is to evaluate the potential contribution of the NGS test in selecting targeted therapies for patients with mBC. Data were retrospectively collected from 101 patients diagnosed with metastatic breast cancer and treated at the Modena Cancer Center between January 2015 and April 2022. A NGS test was performed on the tumor tissue of each patient at the Laboratory of Molecular Pathology of the University Hospital of Modena. This study analyzed the clinical-pathological characteristics and mutational profile of the population using NGS tests, with a focus on actionable mutations that could be targeted in advanced stages of clinical development. The indicator of this study was to quantify the actionable mutations that resulted in a change of cancer treatment. In total, 101 patients with metastatic breast cancer were analyzed, including 86 with luminal phenotype, 10 who were HER2-positive and 5 who were triple-negative. Median age was 52 years. NGS analysis was conducted on 47 samples of primary breast cancer, 52 on metastatic sites of disease and 2 on liquid biopsies. A total of 85 gene mutations were found. The most common mutations were identified in the PIK3CA (47%), FGFR (19%) and ERBB2 genes (12%), and to a lesser extent in other genes. Of the 61 patients with pathogenic mutations, 46 (75%) had at least one actionable mutation. Of these, nine received treatment with a molecular target drug: eight patients with a mutation of the PIK3CA gene were treated with alpelisib and fulvestrant; one patient with FGFR1/2 amplifications received TAS120. Median PFS for these patients was 3.8 months. The study results show that using the NGS test on cancer tissue of metastatic breast cancer could influence the therapeutic choices, considering the small sample size and limited follow-up. About 9% of the study population had their therapy modified based on the results of NGS. The growing number of detectable mutations and increased accessibility of the test may lead to a greater number of potential therapeutic implications for the NGS assay. Perspectives suggest that NGS analysis can be implemented in daily clinical practice, particularly in contexts where a Molecular Tumor Board (MTB) is active.

2023 - Clinicopathological, cytogenetic, and molecular profiles of primary cutaneous diffuse large B-cell lymphomas [Articolo su rivista]
Uccella, Silvia; Goteri, Gaia; Maiorana, Antonino; Donati, Valentina; Tibiletti, Maria Grazia; Magnoli, Francesca; Facchi, Sofia; Merchiori, Deborah; Morsia, Erika; Papotti, Robel; Bettelli, Stefania; Forti, Elisa; Galimberti, Sara; Rupoli, Serena; Filosa, Alessandra; Dardanis, Dimitri; Bomben, Riccardo; Braglia, Luca; Pozzi, Samantha; Sacchi, Stefano
abstract

We analyzed the clinicopathological, cytogenetic, and molecular features of 18 primary cuta-neous diffuse large B-cell lymphomas (PCDLBCLs) and 15 DLBCLs secondarily localized to the skin (SCDLBCLs), highlighting biological similarities and differences between the 2 groups.PCDLBCLs were subclassified after histopathological review as PCDLBCL-leg type (PCDLBCL-LT, 10 cases) and the PCDLBCL-not otherwise specified (PCDLBCL-NOS, 8 cases). Immunohisto-chemistry for Hans' algorithm markers, BCL2, and MYC was performed. The molecular study included the determination of the cell of origin (COO) by Lymph2Cx assay on NanoString platform, FISH analysis of IgH, BCL2, BCL6, and MYC genes, as well as the mutation analysis of MYD88 gene.In immunohistochemistry analysis, BCL2 and MYC hyperexpression was more frequent in LT than in NOS cases and, according to Hans' algorithm, PCDLBCL-LTs were mostly of the non-GC type (8/ 10), whereas in PCDLBCL-NOS, the GC type prevailed (6/8). The determination of COO using Lymph2Cx supported and further confirmed these results. In FISH analysis, all but one LT cases versus 5 of 8 PCDLBCL-NOS showed at least one gene rearrangement among IgH, BCL2, MYC, or BCL6. In addition, MYD88 mutations were more frequently present in LT than in NOS subtypes. Interestingly, MYD88-mutated patients were older, with a non-GC phenotype and had worse OS, compared to MYD88 WT cases. Overall, SCDLBCL did not show, at the genetic and expression level, different pro-files than PCDLBCL, even if they bear a significantly worse prognosis. At survival analysis, the most important prognostic factors in patients with PCDLBCL were age and MYD88 mutation, whereas relapse and high Ki-67 expression were relevant in patients with SCDLBCL.Our study comprehensively analyzed the clinicopathological and molecular features of PCDLBCL-LT, PCDLBCL-NOS, and SCDLBCL, underlining the differences among them and the importance of properly identifying these entities at the time of diagnosis.(c) 2023 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY-NC -ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

2022 - CtDNA-guided rechallenge with anti-EGFR therapy in RASwt metastatic colorectal cancer: Evidence from clinical practice [Articolo su rivista]
D’Onofrio, Raffaella; Caputo, Francesco; Prampolini, Francesco; Spallanzani, Andrea; Gelsomino, Fabio; Bettelli, Stefania; Manfredini, Samantha; Reggiani Bonetti, Luca; Carotenuto, Pietro; Bocconi, Alessandro; Dominici, Massimo; Luppi, Gabriele; Salati, Massimiliano
abstract

Aim: To apply extended ctDNA-based RAS genotyping to clinical criteria for improving the selection of patients eligible for anti-EGFR-based rechallenge in a real-world setting. Methods: ctDNA testing was prospectively applied to RASwt mCRC progressed after a first-line anti-EGFR-containing regimen and at least one other line. The primary endpoint was the objective response rate. Results: Among ten enrolled patients, the anti-EGFR rechallenge resulted in an objective response rate and disease control rate of 70% and 90%. The median progression-free survival was 11.3 months and overall survival was not reached. Compared with a historical cohort retreated with anti-EGFR agents based on clinical criteria, the ctDNA-driven approach resulted in a higher chance of achieving an objective response and longer survival. Conclusions: Blood-based RASwt status may enrich metastatic colorectal cancer more likely to benefit from anti-EGFR-based rechallenge. RAS genotyping in ctDNA represents a feasible, fast, and cost-effective tool to be implemented in the clinic for advancing precision medicine.

2021 - Cell of origin (COO), BCL2/MYC status and IPI define a group of patients with Diffuse Large B-cell Lymphoma (DLBCL) with poor prognosis in a real-world clinical setting [Articolo su rivista]
Bettelli, S.; Marcheselli, R.; Pozzi, S.; Marcheselli, L.; Papotti, R.; Forti, E.; Cox, M. C. C.; Di Napoli, A.; Tadmor, T.; Mansueto, G. R.; Musto, P.; Flenghi, L.; Quintini, M.; Galimberti, S.; Lalinga, V.; Donati, V.; Maiorana, A.; Polliack, A.; Sacchi, S.
abstract

2021 - Deepening the knowledge of ros1 rearrangements in non-small cell lung cancer: Diagnosis, treatment, resistance and concomitant alterations [Articolo su rivista]
Guaitoli, G.; Bertolini, F.; Bettelli, S.; Manfredini, S.; Maur, M.; Trudu, L.; Aramini, B.; Masciale, V.; Grisendi, G.; Dominici, M.; Barbieri, F.
abstract

ROS proto-oncogene 1 (ROS1) rearrangements are reported in about 1–2% of non-squamous non-small-cell lung cancer (NSCLC). After efficacy of crizotinib was demonstrated, identification of ROS1 translocations in advanced disease became fundamental to give patients the chance of specific and effective treatment. Different methods are available for detection of rearrangements, and probably the real prevalence of ROS1 rearrangements is higher than that reported in literature, as our capacity to detect gene rearrangements is improving. In particular, with next generation sequencing (NGS) techniques, we are currently able to assess multiple genes simultaneously with increasing sensitivity. This is leading to overcome the “single oncogenic driver” paradigm, and in the very near future, the co-existence of multiple drivers will probably emerge more frequently and represent a therapeutic issue. Since recently, crizotinib has been the only available therapy, but today, many other tyrosine kinase inhibitors (TKI) are emerging and seem promising both in first and subsequent lines of treatment. Indeed, novel inhibitors are also able to overcome resistance mutations to crizotinib, hypothesizing a possible sequential strategy also in ROS1-rearranged disease. In this review, we will focus on ROS1 rearrangements, dealing with diagnostic aspects, new therapeutic options, resistance issues and the coexistence of ROS1 translocations with other molecular alterations.

2021 - Next Generation Sequencing (NGS): a possible game changer in metastatic breast cancer [Abstract in Rivista]
Barbolini, M.; Omarini, C.; Moscetti, L.; Canino, F.; Trudu, L.; Tornincasa, A.; Caggia, F.; Bettelli, S.; Manfredini, S.; Isca, C.; Molinaro, A.; Dominici, M.; Piacentini, F.
abstract

Background: NGS has been introduced into the clinics with the aim of sequencing long and complex genes for tumor sample, in order to identify driver and/or targetable alterations. Several companies and academic centers have implemented NGS assays to guide treatment decisions, even though there are no clear recommendations from scientific societies about their use in daily clinical practice. Patients and methods: Since 2019 NGS analysis was performed in 32 MBC patients’ tissues at Modena Cancer Center, as for clinical judgement during the course of MBC. Oncomine™ was mainly used for the assay. The aim was to define the PI3K mutational status, since Alpelisib - an a-subunit selective PI3K inhibitor - had shown to improve PFS in PI3K mutated HR+/HER2- MBC patients in SOLAR-1 and BYLIEVE trials. Results: Twenty (62%) NGS analysis were performed on MBC samples, the other (13) on primary breast cancer. Table1 summarize the clinical-pathological characteristics of patients. At least 1 mutation was found in 25 (78%) samples. A PI3K mutation was detected in 14 (44%) cases, with E542K as the most frequent. In 10 out of 14 cases, PI3K mutation was associated with other gene mutations. FGFR3, FGFR4 mutations and FGFR2 amplification were described in 4, 2 and one patients respectively. Two patients showed AKT1 mutation, in one case was associated with PTEN mutation. Two of the patients with PI3K mutation were treated with Alpelisib + Fulvestrant. The patient with FGFR1 amplification was eligible for a phase II clinical trial. Conclusions: NGS performed in this cohort of MBC patients allowed therapeutic decisions in about 10% of cases. Although PI3K mutational status for eligibility to Alpelisib can be cheaply studied by RT-PCR, NGS assay can provide wider information about other gene mutations, useful for patients’ selection for clinical trials. In the era of precision medicine, knowing the mutational status of MBC early in patient history can change the therapeutic algorithm.

2020 - Modulation of Mutational Landscape in HER2-Positive Breast Cancer after Neoadjuvant Chemotherapy [Articolo su rivista]
Omarini, Claudia; Bettelli, Stefania Raffaella; Manfredini, Samantha; Barbolini, Monica; Isca, Chrystel; Cortesi, Giulia; Maiorana, Antonino; Tazzioli, Giovanni; Dominici, Massimo; Piacentini, Federico
abstract

2020 - Overall survival in patients with lung adenocarcinoma harboring "niche" mutations: an observational study [Articolo su rivista]
Aramini, Beatrice; Banchelli, Federico; Bettelli, Stefania Raffaella; Manfredini, Samantha; D'Amico, Roberto; Masciale, Valentina; Pinelli, Massimo; MORETTI FANTERA, Margherita; Stefani, Alessandro; Bertolini, Federica; Dominici, Massimo; Morandi, Uliano; Maiorana, Antonino
abstract

Objective: In addition to the most common somatic lung cancer mutations (i. e., KRAS and EGFR mutations), other genes may harbor mutations that could be relevant for lung cancer. We defined BRAF, c-MET, DDR2, HER2, MAP2K1, NRAS, PIK3CA, and RET mutations as “niche” mutations and analyzed. The aim of this retrospective cohort study was to assess the differences in the overall survival (OS) of patients with lung adenocarcinoma harboring niche somatic mutations. Results: Data were gathered for 252 patients. Mutations were observed in all genes studied, except c-MET, DDR2, MAP2K1, and RET. The multivariable analysis showed that 1) niche mutations had a higher mortality than EGFR mutations (HR = 2.3; 95% CI = 1.2–4.4; p = 0.009); 2) KRAS mutations had a higher mortality than EGFR mutations (HR = 2.5; 95% CI = 1.4–4.5; p = 0.003); 3) niche mutations presented a similar mortality to KRAS mutations (HR = 0.9; 95% CI = 0.6–1.5; p = 0.797). Methods: Three cohorts of mutations were selected from patients with lung adenocarcinoma and their OS was compared. Mutations that were searched for, were 1) BRAF, c-MET, DDR2, HER2, MAP2K1, NRAS, PIK3CA, and RET; 2) K-RAS; and 3) EGFR. Differences in OS between these three cohorts were assessed by means of a multivariable Cox model that adjusted for age, sex, smoking habits, clinical stages, and treatments. Conclusions: Niche mutations exhibited an increased risk of death when compared with EGFR mutations and a similar risk of death when compared with KRAS mutations.

2020 - Predictors of HER2 gene amplification in immunohistochemistry score 2+ Early Breast Cancer according to 2018 ASCO/CAP guidelines: a single institution analysis. [Poster]
Barbolini, M.; Omarini, C.; Bettelli, S.; Manfredini, S.; Dominici, M.; Piacentini, F.
abstract

Background: HER2 overexpression occurs in approximately 15-20% of invasive breast cancers (BC). From a pathological point of view HER2 positivity is defined by intense circumferential membrane complete staining in more than 10% of tumour cells in immunohistochemistry (IHC score 3+). When complete circumferential staining is weak to moderate (IHC score 2+) double probe in situ ibridation (ISH) is mandatory to define HER2 status. In 2018 ASCO/CAP guidelines were updated to provide additional guidance in HER2 equivocal cases to allow a greater discrimination between positive and negative cases. Our aim is to find predictors of HER2 positivity among IHC score 2+ early breast cancer specimens analysed according to 2018 ASCO/CAP guidelines. Patients and methods: 253 cases of early BC diagnosed at Modena Cancer Center between November 2013 and August 2017 were identified. Stage, ISH result, hormonal receptor status (HR), proliferation index (MIB1), and histological grade were captured; menopausal status was available too. All IHC score 2+ cases were reclassified according to 2018 ASCO/CAP guidelines. The association between pathological tumour features, clinical characteristics and ISH positivity was assessed using Fisher test. Results: Overall, 25.7% IHC score 2+ BC resulted HER2 amplified in double probe ISH. High tumour grade (G3 vs G1-2) and MIB1 > 20% significantly predict HER2 ISH amplification (p=0,0001). No correlation was found according to HR, stage, or menopausal status. The majority (185; 98.4%) of HER2-ve BC were reclassified as group 5 (HER2/ CEP17 ratio <2 and HER2 copy number <4 signals/cell) except for 3 specimens classified as group 4 (HER2/CEP17 RATIO <2 and HER2 copy number ³4 but <6 signals/cell). In HER2+ve group the majority (62; 95.3%) specimens were group 1 (HER2/CEP17 RATIO >2 and HER2 copy number =4 signals/cell), no specimen was group 2, and only 3 cases were classified as group 3 (HER2/CEP17 RATIO <2 and HER2 copy number >6 signals/cell). Conclusions: In this IHC score 2+ BC series, reclassification according to 2018 ASCO/CAP guidelines identified only 4.6% group 3 and 1.6% group 4 cases. The routinely assessment of grading and proliferation index could help to predict HER2 amplification in IHC score 2+ samples even if it must not substitute ISH assay in determining eligibility for HER2 targeted therapies.

2020 - Unexpected tumor response to palliative pelvic radiotherapy in mismatch repair-deficient advanced prostate cancer: a case report [Articolo su rivista]
Aluisio, G.; Mazzeo, E.; Lohr, F.; Fiocchi, F.; Bettelli, S.; Baldessari, C.; Paterlini, M.; Bruni, A.
abstract

Background: Mismatch-repair-deficiency resulting in microsatellite instability (MSI) may confer increased radiosensitivity in locally advanced/metastatic tumors and thus radiotherapy (RT) potentially might have a changing role in treating this subset of patients, alone or in combination with checkpoint inhibitors. Case presentation: We report a 76 year-old Italian male patient presenting with locally advanced undifferentiated prostate cancer (LAPC), infiltrating bladder and rectum. Molecular analysis revealed high-MSI with an altered expression of MSH2 and MSH6 at immunohistochemistry. Two months after 6 chemotherapy cycles with Docetaxel associated to an LHRH analogue, a computed tomography scan showed stable disease. After palliative RT (30 Gy/10 fractions) directed to the tumor mass with a 3D-conformal setup, a follow-up computed tomography scan at 8 weeks revealed an impressive response that remained stable at computed tomography after 9 months, with sustained biochemical response. To our knowledge, this is the first case of such a sustained response to low dose RT alone in high-MSI LAPC. Conclusions: Routine evaluation of MSI in patients with locally problematic advanced tumors might change treatment strategy and treatment aim in this setting, from a purely palliative approach to a quasi-curative paradigm.

2019 - Differential molecular pathways expression in HER2 positive early breast cancer according to hormone receptor status. [Articolo su rivista]
Omarini, C; Bettelli, S; Caprera, C; Manfredini, S; Barbolini, M; Moscetti, L; Isca, C; Toss, A; Barbieri, E; Cortesi, L; Kaleci, S; Maiorana, A; Tazzioli, G; Cascinu, S; Piacentini, F
abstract

PURPOSE: Hormone receptors (HR) status in HER2 + breast cancer (BC) is a recognized stratification factor with relevant clinical implication. According to HR expression, HER2 + BC show different clinical characteristics, treatment sensitivity and prognosis. The interaction between HR and HER2 pathways remains incompletely understood. METHODS: Thirty-four HER2 + BC were included: 18 tumors with HER2+/HR + and 16 with HER2+/HR-. The expression of 770 genes and 13 molecular pathways were evaluated using Nanostring PanCancer Pathway panel performed on FFPE BC biopsies. RESULTS: Gene expression analysis identified 127 genes with significantly different expression between the two cohorts. 83% of these genes were overexpressed in HER2+/HR- cohort. Globally, 23% of them belonged to PI3K pathway (41 genes), 15% to Trascriptional regulation (26 genes) and 12% to MAPK (22 genes). Regarding pathway expression, PI3K, MAPK and NOTCH were significantly differently expressed between the two groups (p = 0.003, p = 0.0018 and p = 0.02, respectively), all of them were overexpressed in HER2+/HR- tumors. CONCLUSIONS: According to HR status, HER2 + tumors express different pathways profiles: the overexpression of PI3K, MAPK and NOTCH pathways in HER2+/HR- group could justify different survival outcomes and treatment sensitivity. The identification of tumor driver pathways may be a useful instrument for individualized pathway-directed therapies. Further clinical implications are warranted.

2019 - Quick assessment of cell-free DNA in seminal fluid and fragment size for early non-invasive prostate cancer diagnosis [Articolo su rivista]
Ponti, Giovanni; Maccaferri, Monia; Manfredini, Marco; Micali, Salvatore; Torricelli, Federica; Milandri, Riccardo; Del Prete, Chiara; Ciarrocchi, Alessia; Ruini, Cristel; Benassi, Luisa; Bettelli, Stefania; Kaleci, Shaniko; Ozben, Tomris; Tomasi, Aldo
abstract

Liquid biopsy consists in the quantification and qualification of circulating cell-free DNA (cfDNA) and tumor-derived DNA (ctDNA) for cancer recognition. Recently, the characterization of seminal cfDNA (scfDNA) has been reported as a possible biomarker for prostate cancer (PCa) diagnosis.

2018 - Clinical and molecular predictors of long-term response in HER2 positive metastatic breast cancer patients. [Articolo su rivista]
Omarini, C; Bettelli, S; Caprera, C; Manfredini, S; Caggia, F; Guaitoli, G; Moscetti, L; Toss, A; Cortesi, L; Kaleci, S; Maiorana, A; Cascinu, S; Conte, Pf; Piacentini, F
abstract

BACKGROUND: HER2+ metastatic breast cancer (MBC) is a poor prognosis disease, unusually curable. To date, no predictive factors have been clearly correlated with long-term response to anti-HER2 agents. METHODS: 54 HER2+ MBC patients treated with HER2 targeted therapy as first line treatment were analysed: 40 with a time to progression longer than 3 years in Long Responders (LR) group and 14 with a progression disease within one year of anti-HER2 therapy in a control group named Early Progressors (EP). The expression of 770 genes and 13 molecular pathways were evaluated using Nanostring PanCancer pathway panel performed on FFPE BC tissues. RESULTS: Considering baseline patients and tumor characteristics, EP women had more CNS spread and more metastatic burden of disease compared to LR (p > 0.05). Gene expression analysis identified 30 genes with significantly different expression in the two cohorts; five were driver genes (BRCA1, PDGFRA, AR, PHF6 and MSH2). The majority of these genes were over-expressed, mainly in LR patients, and encoded growth factors, pro- or anti-inflammatory interleukins and DNA repair factors. Only four genes were down regulated, all in EP group (TNFSF10, CACNG1, IL20RB and BRCA1). Most of these genes were involved in MAPK and PI3K pathways. MAPK pathway was differently expressed between LR and EP (p = 0.05). PI3K was the only pathway overexpressed in EP patients. CONCLUSIONS: Whole genome expression analysis comparing LR vs. EP identified a group of genes that may predict more favourable long-term outcomes. Up-regulation of MAPK and down-regulation of PI3K pathway could be a positive predictive factors. Further clinical implications are warranted. ABBREVIATIONS: BC: breast cancer; MBC: metastatic breast cancer; LR: long responder; EP: early progressor; FFPE: formalin-fixed paraffin-embedded; CNS: central nervous system; PFS: progression free survival; OS: overall survival.

2018 - Clinical impact and prognostic role of KRAS/BRAF/PIK3CA mutations in stage I colorectal cancer [Articolo su rivista]
Bonetti, L. R.; Barresi, V.; Maiorana, A.; Manfredini, S.; Caprera, C.; Bettelli, S.
abstract

Stage I colorectal carcinoma has excellent prognosis, with 5-year survival rate up to 95%. The occurrence of lymphovascular invasion, tumor budding, high number of PDC, or lymph node micrometastases is associated with tumor progression. The aim of this study was to evaluate the mutational status of 62 stage I colorectal carcinomas (CRC) (taken from 37 patients surviving more than five years since the initial diagnosis and from 25 patients who died of disease) and to correlate it with histopathological features and the clinical outcome. Mutations of KRAS, NRAS, BRAF, and PIK3CA genes were analyzed through Myriapod Colon Status Kit, using the high-throughput genotyping platform Sequenom MassARRAY System. Mutations in those genes were found in 31 cases (50%) and mainly in those with poor prognosis. The most frequent mutations occurred at codons 12 and 13 of the KRAS gene (40% of cases). We found concomitant PIK3CA mutations in 5 cases (8%). The presence of PIK3CA mutations was mainly observed in tumors with poor prognosis and with unfavorable histopathological prognostic features. High PDC grade (P = 0 0112), the presence of tumor budding (P = 0 0334), LVI (P < 0 0001), KRAS mutations (P = 0 0228), PIK3CA mutations (P = 0 0214), multiple genetic mutations in KRAS and PIK3CA genes (P = 0 039), and nodal micrometastases (P < 0 0001) were significant prognostic variables for CSS. The presence of LVI was the only independent and statistically significant prognostic variable for CSS in our cohort of pTNM stage I CRCs. The analysis of KRAS/PIK3CA mutational status may be used to identify patients with stage I CRC at high risk of bad outcome and who may need additional treatments, including biological therapies.

2018 - Histopathological variables in liver metastases of patients with stage IV colorectal cancer: potential prognostic relevance of poorly differentiated clusters [Articolo su rivista]
Lionti, S.; Reggiani Bonetti, L.; Bettelli, S.; Spallanzani, A.; Gelsomino, F.; Barresi, V.
abstract

The prognosis of patients with colorectal liver metastases (LMs) is mostly established on clinical variables or on the anatomic extent of colorectal cancer (CRC). Histopathological factors of LMs which may actually reflect the biological aggressiveness of the tumor are not routinely considered to define the risk of worse clinical outcome in those patients. The number of poorly differentiated clusters (PDCs) of neoplastic cells in primary CRC is associated with metastatic risk and bad prognosis, but PDC presence in LMs has been barely analyzed thus far. We assessed PDC presence in the histological slides of surgically resected and synchronous LMs in 63 patients with CRC who had been not submitted to any neoadjuvant treatments. Then, we analyzed its association with patients’ cancer-specific survival (CSS) or progression-free survival. The presence of PDCs (P =.016) and PDC localization at tumor edge of LMs (P =.0004) were significantly associated with shorter CSS. PDC presence at the periphery of LMs and positive resection margin were independent prognostic variables for CSS. PDC localization at the tumor edge of LMs was a significant (P =.0079) and independent prognosticator of shorter progression-free survival. Our data suggest that PDC presence and peripheral localization in LMs may be relevant to predict outcome and useful for clinical decision making in patients with colorectal synchronous LMs.

2018 - Mutational Profile of Metastatic Breast Cancer Tissue in Patients Treated with Exemestane Plus Everolimus [Articolo su rivista]
Omarini, Claudia; Filieri, Maria Elisabetta; Bettelli, Stefania Raffaella; Manfredini, Samantha; Kaleci, Shaniko; Caprera, Cecilia; Nasso, Cecilia; Barbolini, Monica; Guaitoli, Giorgia; Moscetti, Luca; Maiorana, Antonino; Conte, Pierfranco; Cascinu, Stefano; Piacentini, Federico
abstract

Background. Everolimus has been shown to overcome endocrine resistance in hormone receptor positive advanced breast cancer patients. Predictive biomarkers of everolimus efficacy have been investigated in primary breast cancer tissue without finding univocal results. The goal of this study was to investigate the mutational burden in the metastatic site of endocrine-resistant tumors treated with everolimus plus exemestane. Patients and Methods. Mass Array Sequenom platform was used to analyse genetic status of 18 cancer-related genes in 25 archival tumor specimens from metastatic lesions and available primary matched breast cancer tissue of patients treated with everolimus and exemestane for advanced disease. An exploratory analysis of everolimus efficacy in terms of progression free survival benefit and single gene mutation was carried out. Results. The overall detection rate of mutation was 30% and 38% from metastatic and primary breast cancer samples, respectively. was the most frequent mutated gene. No primary breast cancer and matched relapse maintained the same mutation profile. Considering molecular pathways, the most of the genes belong to PI3K pathway (, , and ). In patients with detected mutations in breast and/or recurrence tissue the median PFS was 5,6 months while in the subgroup of patients with no mutations the median PFS was 7,5 months. Conclusions. The mutational status of breast cancer recurrence allows the identification of some genes potentially correlating tumor response/resistance to everolimus. The most frequently mutated genes were involved in the PI3K/AKT/mTOR pathway highlighting that the deregulation of this pathway in the relapse plays a crucial role in the mechanisms of everolimus resistance/sensitivity. Owing to the small sample size and the retrospective nature of the study, these correlations need to be validated in a large prospective study.

2017 - Analysis of KRAS, NRAS, PIK3CA, and BRAF mutational profile in poorly differentiated clusters of KRAS-mutated colon cancer [Articolo su rivista]
Reggiani Bonetti, Luca; Barresi, Valeria; Bettelli, Stefania; Caprera, Cecilia; Manfredini, Samantha; Maiorana, Antonio
abstract

Recently, a grading system based on the counting of poorly differentiated clusters (PDCs) of neoplastic cells was shown to be a strong predictor of nodal metastases and negative prognosis in colon cancer (CC). In this study, we assessed and compared the mutational status of KRAS, NRAS, and PIK3CA in PDCs and corresponding main tumor tissue of 25 CCs with KRAS mutations. For each tumor, PDC and main tumor tissue were distinctly analyzed by using laser microdissection and mass spectrometry. In 3 CCs, the main tumor tissue had also PIK3CA mutations (C420R: 1; E545K: 1; H1047R: 1), and in 1, it showed NRAS mutation (codon 12). In 20 cases, PDCs had the same biomolecular profile as the main tumor, but in 5, they had different biomolecular profiles. In detail, PDCs had KRAS wild type in 2 cases and additional PIK3CA mutations (E542K: 1; H1047Y: 1; E545Q: 1) in 3. All 3 cases with additional PIK3CA mutations in PDCs had nodal metastases, high pathological TNM stage, and lymphatic invasion. In 1 of 3 cases, additional PIK3CA mutation detected in PDC, but not in the main tumor, was also found in the corresponding nodal metastases. Our findings show for the first time that heterogeneous biomolecular profile previously observed in CC may depend on different histologic aspects of the lesion. Because PDCs may represent the tumor cells with the highest potential to metastatize, their molecular status may be relevant for the prediction of response to targeted therapies.

2017 - Differential gene expression patterns in HER2 positive metastatic breast cancer patients according to hormone receptor status [Poster]
Omarini, Claudia; Kaleci, Shaniko; Guaitoli, Giorgia; Bettelli, Stefania Raffaella; Cecilia, Caprera; Manfredini, Samantha; Caggia, Federica; Baschieri, MARIA CRISTINA; Luca, Moscetti; Maiorana, Antonino; Cascinu, Stefano; Piacentini, Federico
abstract

Background: HER2 positive breast cancer (HER2+ BC) is a heterogeneous disease. Presenting features, patterns of recurrence and survival of HER2+ BC can differ according to hormone receptors (HR) status. The purpose of this study is to highlight different gene profiles and molecular pathways between HR+ and HR- metastatic HER2+ BCs. Material and Methods: 34 HER2+ metastatic BC patients were included: 18 patients with HR+/HER2+ and 14 with HR-/HER2+. Data regarding tumor characteristics, treatment information and clinical outcomes were collected. The expression of 770 genes and 13 molecular pathways were evaluated by means of Nanostring PanCancer pathway panel performed on BC formalin-fixed paraffin-embedded tissues from diagnostic core biopsy or surgical resection. Results: Gene expression analysis identified 118 genes with significantly different expression in the two cohorts. All but one of these genes were over-expressed; only the gene CACNG6 was down-regulated in HR+/HER2+ group. In particular, 93 genes were over-expressed in HR-/HER2+ while 24 were overexpressed in HR+/HER2+. Most of these genes encoded growth factors, pro- or anti-inflammatory interleukins and DNA repair factors. 62% of these genes were involved in PI3K, MAPK and RAS pathways (32, 22 and 18, respectively). PI3K, MAPK and NOTCH pathways were differently expressed between HR+/HER2+ and HR-/HER2+ (p=0.003, p=0.0018, p=0.02, respectively). All these three pathways were overexpressed in HR-/HER2+ BC. In particular, all the significantly different expression genes in NOTCH pathways were overexpressed in HR-/HER2+ group. Conclusions: This genome expression analysis identified a gene expression profile able to differentiate HR+ versus HR- HER2+ metastatic BC. The overexpression of PI3K, MAPK and NOTCH pathways in HR-/HER2+ BC could justify its more aggressive behaviour. The validation of this HER2+ BC profile needs further investigation. Keywords: metastatic breast cancer, gene expression profile, HER2 positive breast cancer, PanCancer

2017 - Differential gene expression patterns in HER2 positive metastatic breast cancer patients according to hormone receptor status. [Abstract in Rivista]
Omarini, C; Kaleci, S; Guaitoli, G; Bettelli, S; Caprera, Cecilia; Manfredini, S; Caggia, F; Baschieri, Cristina; Moscetti, Luca; Maiorana, A; Cascinu, S; Piacentini, F
abstract

Background: HER2 positive breast cancer (HER2+ BC) is a heterogeneous disease. Presenting features, patterns of recurrence and survival of HER2+ BC can differ according to hormone receptors (HR) status. The purpose of this study is to highlight different gene profile and molecular pathways between HR+ and HR- metastatic HER2+ BCs. Materials and methods: 34 HER2+ metastatic BC patients were included: 18 patients with HR+/HER2+ and 14 with HR-/HER2+. Data regarding tumor characteristics, treatment information and clinical outcomes were collected. The expression of 770 genes and 13 molecular pathways were evaluated by means of Nanostring PanCancer pathway panel performed on BC formalin-fixed paraffin-embedded tissues from diagnostic core biopsy or surgical resection specimen. Results: Gene expression analysis identified 118 genes with significantly different expression in the two cohorts. All but one of these genes were over-expressed; only the gene CACNG6 was down-regulated in HR+/HER2+ group. In particular, 93 genes were over-expressed in HR-/HER2+ while 24 were overexpressed in HR+/HER2+. Most of these genes encoded growth factors, pro- or anti-inflammatory interleukins and DNA repair factors. 62% of these genes were involved in PI3K, MAPK and RAS pathways (32, 22 and 18, respectively). PI3K, MAPK and NOTCH pathways were differently expressed between HR+/HER2+ and HR-/HER2 + (p = 0.003, p = 0.0018, p = 0.02, respectively). All these three pathways were overexpressed in HR-/HER2+ BC. In particular, all the significantly different expression genes in NOTCH pathways were overexpressed in HR-/HER2+ group. Conclusions: This genome expression analysis identified a gene expression profile able to differentiate HR+ versus HR- HER2+ metastatic BC. The overexpression of PI3K, MAPK and NOTCH pathways in HR-/HER2+ BC could justify its more aggressive behaviour. The validation of this HER2+ BC profile needs further investigation.

2017 - Durable remission in a patient with leptomeningeal relapse of a MYC/BCL6-positive double-hit DLBCL treated with lenalidomide monotherapy [Articolo su rivista]
Salati, Massimiliano; Tarantino, Vittoria; Maiorana, Antonino; Bettelli, Stefania Raffaella; Luminari, Stefano
abstract

Secondary central nervous system involvement is an uncommon event that typically occurs early in the natural history of diffuse large B-cell lymphoma and presents as leptomeningeal dissemination in two-thirds of cases. The prognosis of this event is dismal, and treatment options are meagre. Although major validated risk factors for central nervous system dissemination are clinical, concomitant MYC/BCL2 rearrangements as well as MYC/BCL2 protein expression have been recently associated with an increased risk of this complication. Here we present the first case, to our knowledge, of a MYC/BCL6-positive double-hit diffuse large B-cell lymphoma relapsing in the leptomeninges that achieved an outstanding durable remission with single-agent lenalidomide following salvage chemotherapy.

2016 - Clinical and molecular analysis of long-term HER2 positive metastatic breast cancer survivors. [Poster]
Omarini, Claudia; Caprera, Cecilia; Manfredini, Samantha; Caggia, Federica; Guaitoli, Giorgia; Filieri, Maria Elisabetta; Moscetti, Luca; Bettelli, Stefania Raffaella; Kaleci, Shaniko; Cascinu, Stefano; Piacentini, Federico
abstract

ackground: Several multigene tests have been developed in Metastatic Breast Cancer (MBC) disease, in order to identify predictive factors correlated to clinical outcomes. The purpose of this study is to investigate the clinico-pathological and molecular characteristics that could differentiate long term responders from patients experiencing early progression during anti-HER2 treatments. Methods: A total of 34 HER2 positive MBC patients were included: 20 patients with a time to progression longer than 3 years in Long Responders group (LR) and 14 patients with a progression disease within one year of anti-HER2 therapy in Poor Responders group (PR). Tumor characteristics and treatment information were collected. The expression of 770 genes and 13 molecular pathways were evaluated using Nanostring PanCancer pathway panel performed on BC formalin-fixed paraffin-embedded tissues from diagnostic core biopsy or surgical resection. Results: Baseline patients and tumor characteristics were similar between the two groups, although PR patients were more likely to have CNS spread and more metastatic burden of disease compared to LR (29% vs. 0, p = 0.02 and 57% vs. 20%, p = 0.04, respectively). Gene expression analysis identified 30 genes with significantly different expression in the two cohorts; five of these were driver genes (BRCA1, PDGFRA, AR, PHF6 and MSH2). The majority of these genes were over-expressed, mainly in LR patients, and encoded growth factors, pro- or anti-inflammatory interleukins and DNA repair factors. Only four genes were down-regulated, all in PR group (TNFSF10, CACNG1, IL20RB and BRCA1). Most of these genes were involved in MAPK and PI3K pathways (9 and 8, respectively). MAPK pathway was differently expressed between LR and PR (p = 0.05). Even if not statistically significant but clinically relevant, PI3K was the only pathway overexpressed in PR patients (median expression LR: 1441 ± 485 vs 1759 ± 762 in PR group; p= 0.1). Conclusions: Whole genome expression analysis comparing LR vs. PR identified a group of genes that may predict more favourable long-term outcomes. Up-regulation of MAPK and down-regulation of PI3K pathways could be a positive predictive factors. Further clinical implications are warranted.

2016 - Molecular profile in primary and metastatic breast cancer treated with Exemestane and Everolimus. [Abstract in Atti di Convegno]
Filieri, Maria Elisabetta; Bettelli, Stefania Raffaella; Maiorana, Antonio; Caprera, Cecilia; Manfredini, Samantha; Caggia, Federica; Iattoni, Elena; Cascinu, Stefano; Omarini, Claudia; Piacentini, Federico
abstract

Background. The PI3K/Akt/ mTOR pathway plays a significant role in endocrine resistance breast cancer (BC). Everolimus (EVE) has been approved after BOLERO-2 study, which showed a significant increase of PFS thanks to EVE plus Exemestane (EXE), compared to EXE alone. Hortobagyi et al. performed Next Generation Sequencing on 227 BOLERO-2 samples of primary BC to study the potential correlation between genetic alterations and EVE efficacy: a greater incidence of mutations in PI3KCA, PTEN, CCND1 and FGFR1 was detected, but treatment effect was independent from the genetic status. The aim of this study was to evaluate the molecular profile of both primary and secondary lesions in 25 metastatic BC patients treated with EVE+EXE in Modena University Hospital since 2014. Materials and methods. Thirty-three DNA samples from 25 patients were examined, 13 from primary BC and 20 from metastatic lesions. In 8 patients both primary tumor and metachronous metastasis were evaluated. Genomic DNA samples from FFPE tissues was conducted using panel OncoCarta 2.0 on MassArray Sequenom platform that detect more than 150 single nucleotide variations from 18 genes (AKT1, BRAF, CTNNB1, FBX4, FBXW7, FGFR2/3, GNAQ, KIT, KRAS, MAP2K1/2, NRAS, PDGFR, PIK3CA, PTPN11, SOS1, TP53). Differences between mutational status of primary and metastatic BC have been evaluated using χ2 and Fisher tests. Results. The median age was 54 years (range 50-67). 70% of patients had visceral involvement, 62% received more than 3 previous therapies, and for 8% of them an AI constituted the last treatment before EVE. Overall, 11 DNA samples were mutated (33%). 5 mutations were detected in the primary lesion with a frequency of 3% (PI3KCA, FBX4, KIT, MAP2K1, FBXW7) - 6 mutations in the metastasis (BRAF, KIT, TP53, FBXW7, CTNNB1, PI3KCA, AKT). Notably, mutations were found exclusively in primary lesion or in metastatic site, while only in one case both primary and secondary cancer were mutated, even if in two different genes. No significant correlation with treatment efficacy was evidenced. Conclusions. The genes most frequently mutated in MBC were PI3KCA, AKT1 and FBXW7, even if the percentage of PIK3CA and AKT1 mutations was less than expected. No correlation between primary and metastatic mutational status was detected. Involving new patients maybe could be the way for more encouraging results.

2016 - Poorly differentiated clusters (PDC) in colorectal cancer: What is and ought to be known. [Articolo su rivista]
Reggiani Bonetti, Luca; Barresi, Valeria; Bettelli, Stefania; Domati, Federica; Palmiere, Cristian
abstract

Background: The counting of poorly differentiated clusters of 5 or more cancer cells lacking a gland-like structure in a tumor mass has recently been identified among the histological features predictive of poor prognosis in colorectal cancer. Main body: Poorly differentiated clusters can easily be recognized in the histological sections of colorectal cancer routinely stained with haematoxylin and eosin. Despite some limitations related to specimen fragmentation, counting can also be assessed in endoscopic biopsies. Based on the number of poorly differentiated clusters that appear under a microscopic field of a ×20 objective lens (i.e., a microscopic field with a major axis of 1 mm), colorectal cancer can be graded into malignancies as follows: tumors with <5 clusters as grade 1, tumors with 5 to 9 clusters as grade 2, and tumors with ≥10 clusters as grade 3. High poorly differentiated cluster counts are significantly associated with peri-neural and lympho-vascular invasion, the presence of nodal metastases or micrometastases, as well as shorter overall and progression free survival to colorectal cancer. Conclusion: The morphological aspects and clinical relevance of poorly differentiated clusters counting in colorectal cancer are discussed in this review.

2016 - Prediction of Distant Recurrence-Free Survival in Resectable Lung Adenocarcinoma. [Abstract in Atti di Convegno]
Aramini, B; Casali, C; Stefani, A; Bettelli, S; Wagner, ; Sangale, Z; Hughes, E; S., Lanchbury J; Maiorana, A; Morandi, U.
abstract

OBJECTIVES: Optimal procedures for adjuvant treatment and post-surgical surveillance of resected non-small-cell lung cancer remain under discussion. Pathological features are the main determinant of follow-up therapy but have limited ability to identify patients at risk of recurrence. Increasingly, molecular markers are incorporated into clinical decision-making, including measures of tumor growth. The CCP score is a quantitative, molecular measure of proliferation derived from the RNA expression of 31 cell cycle genes and a component of the molecular prognostic score (mPS). The mPS score is a linear combination of CCP score and pathological stage. CCP score and mPS are independent predictors of survival in resected lung adenocarcinoma. MATERIALS AND METHODS: CCP scores were determined by RT-qPCR for 318 patients diagnosed with stage I-II lung adenocarcinoma. Association of mPS and CCP score with distant recurrence and lung-cancer specific survival was assessed in Cox proportional hazards regression models adjusted for age, gender, tumor size, pathological stage and pleural invasion. Distant recurrence-free survival and lung-cancer specific survival by mPS risk group were calculated by Kaplan-Meier survival analysis. RESULTS: CCP scores were obtained for 205 stage I and 84 stage II patients. CCP score and mPS were independent markers of distant recurrence (CCP: HR 1.62, 95%CI 1.15-2.29, p=0.0055; mPS: HR 2.22, 95%CI 1.11-4.44, p=0.023). Patients with low mPS tumors were at significantly reduced risk of distant recurrence (log-rank p=4.2×10-5). Among stage I patients, stratification by mPS identified a patient group with increased risk of distant recurrence (36%, 95%CI 28-46%, log-rank p=0.0011) CONCLUSIONS: The molecular prognostic score stratifies early-stage, resected lung cancer patients for risk of distant recurrence and could be useful to inform treatment and surveillance decisions.

2016 - Prediction of distant recurrence in resected stage I and II lung adenocarcinoma [Articolo su rivista]
Aramini, Beatrice; Casali, Christian; Stefani, Alessandro; Bettelli, Stefania; Wagner, Susanne; Sangale, Zaina; Hughes, Elisha; Lanchbury, Jerry S.; Maiorana, Antonino; Morandi, Uliano
abstract

Objectives Optimal procedures for adjuvant treatment and post-surgical surveillance of resected non-small-cell lung cancer remain under discussion. Pathological features are the main determinant of follow-up therapy but have limited ability to identify patients at risk of recurrence. Increasingly, molecular markers are incorporated into clinical decision-making, including measures of tumor growth. The CCP score is a quantitative, molecular measure of proliferation derived from the RNA expression of 31 cell cycle genes and a component of the molecular prognostic score (mPS). The mPS score is a linear combination of CCP score and pathological stage. CCP score and mPS are independent predictors of survival in resected lung adenocarcinoma. Materials and methods CCP scores were determined by RT-qPCR for 318 patients diagnosed with stage I–II lung adenocarcinoma. Association of mPS and CCP score with distant recurrence and lung-cancer specific survival was assessed in Cox proportional hazards regression models adjusted for age, gender, tumor size, pathological stage and pleural invasion. Distant recurrence-free survival and lung-cancer specific survival by mPS risk group were calculated by Kaplan-Meier survival analysis. Results CCP scores were obtained for 205 stage I and 84 stage II patients. CCP score and mPS were independent markers of distant recurrence (CCP: HR 1.62, 95%CI 1.15-2.29, p = 0.0055; mPS: HR 2.22, 95%CI 1.11-4.44, p = 0.023). Patients with low mPS tumors were at significantly reduced risk of distant recurrence (log-rank p = 4.2 × 10−5). Among stage I patients, stratification by mPS identified a patient group with increased risk of distant recurrence (36%, 95%CI 28–46%, log-rank p = 0.0011) Conclusions The molecular prognostic score stratifies early-stage, resected lung cancer patients for risk of distant recurrence and could be useful to inform treatment and surveillance decisions.

2015 - Assessment of freezing effects and diagnostic potential of BioBank healthy and neoplastic breast tissues through HR-MAS NMR spectroscopy [Articolo su rivista]
Righi, Valeria; Schenetti, Luisa; Maiorana, Antonino; Libertini, Emanuela; Bettelli, Stefania Raffaella; Reggiani Bonetti, Luca; Mucci, Adele
abstract

HR-MAS NMR spectroscopy was employed to monitor the metabolic profiles of Modena BioBank breast samples over 1 year of freezing at -80 C. The study includes 22 adult female patients living in Modena and its hinterland, who underwent total mastectomy or quadrantectomy in 2011–2012. Variations occur, especially affecting phosphocholine (PC) and choline. This is not a trivial finding, since many studies base the distinction between neoplastic and healthy tissues or the assessment of tumor grade on the analysis of choline metabolites. Despite the changes observed, we established that the diagnostic power of the HR-MAS NMR spectra of frozen samples is preserved, at least as far as the distinction between neoplastic and healthy samples is concerned. Lactate (Lac), PC, phosphoethanolamine (PE), taurine (Tau), myo-inositol (Myo) and glucose (Glc) are biomarkers that can be used to distinguish healthy from neoplastic tissues, whereas some metabolite ratios, such as Lac ? PE ? Tau/Glc ? Myo, seem to have even higher discrimination potential.

2015 - High-Resolution Melting is a sensitive, cost-effective, time-saving technique for BRAF V600E detection in thyroid FNAB washing fluid: a prospective cohort study [Articolo su rivista]
Marino, Marco; Monzani, Maria Laura; Brigante, Giulia; Cioni, Katia; Madeo, Bruno; Santi, Daniele; Maiorana, Antonino; Bettelli, Stefania Raffaella; Moriondo, Valeria; Pignatti, Elisa; Bonacini, Lara; Carani, Cesare; Rochira, Vincenzo; Simoni, Manuela
abstract

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2015 - KRAS, NRAS, BRAF mutations and high counts of poorly differentiated clusters of neoplastic cells in colorectal cancer: Observational analysis of 175 cases [Articolo su rivista]
Barresi, Valeria; Bonetti, Luca Reggiani; Bettelli, Stefania
abstract

A novel grading system based on the counting of poorly differentiated clusters (PDC) of neoplastic cells at the invasive margin and in the tumour stroma was recently introduced among the histological parameters predictive of adverse clinical outcome in colorectal cancer (CRC). The aim of this study was to correlate the histological grade based on PDC and the mutational status of KRAS, NRAS and BRAF genes in 175 consecutive CRCs. The highest PDC count under the objective lens of a x20 microscopic field in each tumour was considered for grading assessment, so that PDC counts <5, 5-9 and ≥10 PDCs were defined grade 1, grade 2 and grade 3, respectively. Hotspots mutations were identified using the MassArray platform. Overall, there were 42 (24%) mutated tumours. Mutational status was significantly associated with high pT stage (p = 0.0021), advanced pTNM stage (p = 0.0018), nodal metastases (p = 0.006), tumour budding (p = 0.022) and high PDC grade (p = 0.0022). KRAS mutations were significantly associated with PDC grade (p = 0.0379), while BRAF mutations were associated with PDC-G3 although statistical significance was not reached. No significant associations were found between NRAS and PDC. The significant association between mutated KRAS and PDC grade suggests that KRAS mutations may be involved in the formation of PDC.

2015 - Molecular Profile, as detected with Mass-Array Spectrometry (Sequenom platform), in primary and metastatic breast carcinoma treated with Exemestane + Everolimus [Abstract in Atti di Convegno]
Manfredini, S.; Bettelli, S.; Filieri, M. E.; Caprera, C.; Ficarra, G.; Piacentini, F.; Maiorana, A.
abstract

Background. PI3K/Akt/mTOR is one of the most important pathways for the regulation of cell survival, proliferation and apoptosis. Mutational events occurring in this pathway could lead to malignant transformation and endocrine resistance in breast cancer. The mTOR inhibitor Everolimus (EVE) interferes with cellular proliferation by binding FKB12 protein. EVE has been definitely approved thanks to BOLERO-2 phase III study, which showed a significant prolongation of Progression Free Survival (PFS) due to the addiction of EVE to Exemestane therapy, compared to Exemestane alone, in Hormonal Receptors-positive (RO+) and HER2-negative (HER2-) metastatic breast cancer patients. Hortobagyi et al. performed Next Generation Sequencing on 227 BOLERO-2 samples of primary breast carcinoma, to study the potential correlation between genetic alterations and EVE efficacy. A greater incidence of mutations in PIK3CA, PTEN, CCND1 and FGFR1/2 genes was detected and it was observed that patients with no or only one genetic alteration in these genes derive the most benefit from EVE therapy. To our knowledge, no previous research has evaluated the mutational status both in primary and metastatic breast cancers. Materials and methods. Aim of this study was to evaluate the molecular profile in primitive breast cancers (21 ductal carcinomas, 3 lobular carcinomas and 1 colloid carcinoma) and visceral metastases (hepatic and pulmonary), in 25 patients with advanced breast cancer (RO+ HER2-) treated with EVE in combination with Exemestane. Thirty-three DNA samples from 25 patients were examined, 13 from primary0 breast cancers and 20 from metastatic lesions. In 8 patients, both the primary tumor and the corresponding metachronous metastasis were evaluated. Genomic DNA samples from FFPE tumoral tissue were analized by using OncoCarta v2.0 panel on Mass Array Sequenom platform. A preliminar Multiplex-PCR, followed by SAP-dephosphorylating reaction and iPLEX-primer specific extension, was performed to detect more than 150 single nucleotide variations in mutational hotspots from 18 implicated genes (AKT1, BRAF, CTNNB1, FBX4, FBXW7, FGFR2, FGFR3, GNAQ, KIT, KRAS, MAP2K1, MAP2K2, NRAS, PDGFRa, PIK3CA, PTPN11, SOS1, TP53). Differences were evaluated using Chi-Square and Fisher Tests. Survival analysis was conducted using Kaplan-Meyer curves. Results. Overall, 11 DNA samples, out of the 33 examined, were mutated (33%). Mutations were found in 10 ductal carcinomas and in the colloid carcinoma. Five mutations were detected in primary breast lesions and 6 in metastatic ones. All mutations consisted of a single-nucleotide variation resulting in aminoacidic substitution. Among primary lesions, mutations were detected in the following genes: PIK3CA (E545K), FBX4 (G30N), KIT (S709F), MAP2K1 (D67N), FBXW7 (R465C). They occurred with a frequency of 3%, respectively, namely in 1 out of 33 samples each. Only in the AKT1 gene the same mutation (E17K) was found in 2 DNA primary lesion samples. In metastatic lesions, BRAF (R444W), KIT (G565R), TP53 (R273H), FBXW7 (R479Q), CTNNB1 (S45F), PIK3CA (E545K), AKT (E17K) were mutated. Notably, mutations were found exclusively in primary lesions or in metastatic ones, while only in one patient both primary and secondary lesions were mutated; however, these mutations occurred in two different genes: MAP2K1 (D67N) in breast, FBXW7 (R479Q) in metastasis. Of notice, a reduction in PFS was observed in one patient which carried 3 different mutations (FBX4, PIK3CA, KIT) in the primary tumor (3.4 month versus an average of 5 month) whereas a significantly increased PFS (15.9 month) was detected in a case with 2 mutations (PIK3CA, AKT1) in metastatic lesion. Conclusion. Although the number of patients and samples is quite limited, our findings in mutational status support literature evidence, as genes most frequently mutated were PIK3CA, AKT1 and FBXW7, even if the percentage of

2015 - Prospective biomarker analysis of the randomized CHER-LOB study evaluating the dual anti-HER2 treatment with trastuzumab and lapatinib plus chemotherapy as neoadjuvant therapy for HER2-positive breast cancer [Articolo su rivista]
Guarneri, Valentina; Dieci, Maria Vittoria; Frassoldati, Antonio; Maiorana, Antonino; Ficarra, Guido; Bettelli, Stefania Raffaella; Tagliafico, Enrico; Bicciato, Silvio; Generali, Daniele Giulio; Cagossi, Katia; Bisagni, Giancarlo; Sarti, Samanta; Musolino, Antonino; Ellis, Catherine; Crescenzo, Rocco; Conte, Pierfranco
abstract

Background. The CHER-LOB randomized phase II study showed that the combination of lapatinib and trastuzumab plus chemotherapy increases the pathologic complete re- mission (pCR) rate compared with chemotherapy plus either trastuzumab or lapatinib. A biomarker program was prospectively planned to identify potential predictors of sensitivity to different treatments and to evaluate treatment effect on tumor biomarkers. Materials and Methods. Overall, 121 breast cancer patients positive for human epidermal growth factor 2 (HER2) were randomly assigned to neoadjuvant chemotherapy plus trastu- zumab, lapatinib, or both trastuzumab and lapatinib. Pre-and post-treatment samples were centrally evaluated for HER2, p95- HER2, phosphorylated AKT (pAKT), phosphatase and tensin homolog, Ki67, apoptosis, and PIK3CA mutations. Fresh-frozen tissue samples were collected for genomic analyses. Results. A mutation in PIK3CA exon 20 or 9 was documented in 20% of cases. Overall, the pCR rates were similar in PIK3CA wild- type and PIK3CA-mutated patients (33.3% vs. 22.7%; p 5.323). For patients receiving trastuzumab plus lapatinib, the probabil- ity of pCR was higher in PIK3CA wild-type tumors (48.4% vs. 12.5%; p 5.06). Ki67, pAKT, and apoptosis measured on the residual disease were significantly reduced from baseline. The degree of Ki67 inhibition was significantly higher in patients receiving the dual anti-HER2 blockade.The integrated analysis of gene expression and copy number data demonstrated that a 50- gene signature specifically predicted the lapatinib-induced pCR. Conclusion. PIK3CA mutations seem to identify patients who are less likely to benefit from dual anti-HER2 inhibition. p95-HER2 and markers of phosphoinositide 3-kinase pathway deregulation are not confirmed as markers of different sensitivity to trastuzumab or lapatinib.

2014 - ASSESSMENT OF FREEZING EFFECTS AND DIAGNOSTIC POTENTIAL OF BIOBANK HEALTHY AND NEOPLASTIC BREAST TISSUES THROUGH HR-MAS NMR SPECTROSCOPY [Abstract in Atti di Convegno]
Mucci, Adele; Schenetti, Luisa; Maiorana, Antonino; Bettelli, Stefania Raffaella; REGGIANI BONETTI, Luca; V., Righi
abstract

HR-MAS NMR spectroscopy was employed to monitor the metabolic profiles of Modena BioBank breast samples over one year of freezing at -80 °C. The study includes 22 adult female patients living in Modena and its hinterland, who underwent total mastectomy or quadrantectomy in 2011 - 2012. Variations occur, especially affecting phosphocholine and choline. This is not a trivial finding, since many studies base the distinction between neoplastic and healthy tissues or the assessment of tumor grade on the analysis of choline metabolites [1,2]. Despite the changes observed, we established that the diagnostic power of the HR-MAS NMR spectra of frozen samples is preserved, at least as far as the distinction between neoplastic and healthy samples is concerned. Lactate, phosphocholine, phosphoethanolamine, taurine, myo-inositol and glucose are biomarkers that can be used to distinguish healthy from neoplastic tissues, whereas some metabolite ratios, such as Lac+PE+Tau/Glc+Myo, seem to have even higher discrimination potential. Fig. 1. Scores plot of PCA on neoplastic (crosses) vs healthy (triangles) samples. Loadings profiles (right) of PC2 and PC3. References [1]

2014 - Double-blind, placebo-controlled, multicenter, randomized, phase IIb neoadjuvant study of letrozole-lapatinib in postmenopausal hormone receptor-positive, human epidermal growth factor receptor 2-negative, operable breast cancer [Articolo su rivista]
Guarneri, Valentina; Generali, Daniele Giulio; Frassoldati, Antonio; Artioli, Fabrizio; Boni, Corrado; Cavanna, Luigi; Tagliafico, Enrico; Maiorana, Antonino; Bottini, Alberto; Cagossi, Katia; Bisagni, Giancarlo; Piacentini, Federico; Ficarra, Guido; Bettelli, Stefania Raffaella; Roncaglia, Enrica; Nuzzo, Simona; Swaby, Ramona; Ellis, Catherine; Holford, Clare; Conte, Pierfranco
abstract

Purpose This is a randomized, double-blind, placebo-controlled study aimed to evaluate the clinical and biologic effects of letrozole plus lapatinib or placebo as neoadjuvant therapy in hormone receptor (HR) –positive/human epidermal growth factor receptor 2 (HER2) –negative operable breast cancer. Methods Ninety-two postmenopausal women with stage II to IIIA primary breast cancer were randomly assigned to preoperative therapy consisting of 6 months of letrozole 2.5 mg orally daily plus lapatinib 1,500 mg orally daily or placebo. Surgery was performed within 2 weeks from the last study medication. Clinical response was assessed by ultrasonography. Pre- and post-treatment samples were evaluated for selected biomarkers. Fresh-frozen tissue samples were collected for genomic analyses. Results Numerically similar clinical response rates (partial complete response) were observed (70% for letrozole-lapatinib and 63% for letrozole-placebo). Toxicities were generally mild and manageable. A significant decrease in Ki-67 and pAKT expression from baseline to surgery was observed in both arms. Overall, 34 patients (37%) had a mutation in PIK3CA exon 9 or 20. In the letrozole-lapatinib arm, the probability of achieving a clinical response was significantly higher in the presence of PIK3CA mutation (objective response rate, 93% v 63% in PIK3CA wild type; P .040). Conclusion The combination of letrozole-lapatinib in early breast cancer was feasible, with expected and manageable toxicities. In unselected estrogen receptor–positive/HER2-negative patients, letrozolelapatinib and letrozole-placebo resulted in a similar overall clinical response rate and similar effect on Ki-67 and pAKT. Our secondary end point findings of a significant correlation between PIK3CA mutation and response to letrozole-lapatinib in HR-positive/HER2-negative early breast cancer must now be independently confirmed.

2013 - Evaluation of the effects of cryopreservation on Modena Biobank tissue samples through HR-MAS NMR [Abstract in Atti di Convegno]
Valeria, Righi; Schenetti, Luisa; Maiorana, Antonino; Bettelli, Stefania Raffaella; REGGIANI BONETTI, Luca; Mucci, Adele
abstract

The application of high-resolution magic angle spinning (HR-MAS) NMR spectroscopy to the analysis of intact tissue biopsies and surgery samples dates back to 1997. It represents, at present, an actively investigated field, and it is used to obtain the metabolic fingerprints of tissues. It has been shown to differentiate between normal and neoplastic tissue in the case of breast, brain, kidney, colon cancer and other malignancies in the upper gastrointestinal tract. Nevertheless, it is difficult to run HR-MAS analysis immediately after surgical resection or biopsy, and samples are currently frozen in liquid nitrogen and then stored to -80 °C. Some studies reporting the effect of sample ischaemia, spinning time and measurement temperature on the metabolic profile of tissues can be found.1 Nevertheless, another important issue to be addressed is the effect of cryopreservation time on the metabolic profile of tissues. Crypreservation represents a more and more common practice, due to the widespread of tissue BioBanks. To gain a deeper insight into metabolic changes that occur during the storage, samples of Modena BioBank were analyzed through HR-MAS NMR after 1, 6 and 12 months from collection. The results of this study were discussed.

2013 - Loss of HER2 positivity and prognosis after neoadjuvant therapy in HER2-positive breast cancer patients [Articolo su rivista]
Guarneri, Valentina; Dieci, Maria Vittoria; Barbieri, E; Piacentini, Federico; Omarini, Claudia; Ficarra, G; Bettelli, Stefania Raffaella; Conte, Pierfranco
abstract

Background: Emerging literature data are showing that a change in human epidermal growth factor receptor (HER2) status adversely affects breast cancer patient’s prognosis. The aim of this study was to evaluate the prognostic impact of HER2 loss in patients with HER2-positive disease treated with neoadjuvant therapy with or without anti-HER2 agents. Methods: One hundred and seven consecutive HER2-positive patients were identified from a prospectively maintained database. The first cohort includes 40 patients treated with chemotherapy (CT) alone. The second cohort includes 67 patients treated with neoadjuvant CT plus anti-HER2 agents (trastuzumab and/or lapatinib). HER2 expression was evaluated by immunihistochemistry or fluorescence in situ hybridization on pretreatment core biopsy and on surgical specimen after therapy. Results: The rates of pathologic complete response (pCR) and breast-conserving surgery were higher in the CT + anti- HER2 cohort. A loss of HER2 expression was observed in 40% of the patients with residual disease after CT alone versus 14.7% of the patients after CT + anti-HER2 agents (P = 0.019). Patients not achieving a pCR have a significant increase in the risk of relapse when compared with those achieving a pCR (hazard ratio [HR] 9.55, P = 0.028). Patients with HER2 loss tended to have a higher risk of relapse as comparing to patients with maintained HER2 positivity (HR 2.41, P = 0.063). Conclusion: The pCR is confirmed as a powerful predictor of long-term outcome. The rate of HER2 loss is higher in patients receiving neoadjuvant CT without anti-HER2 agents. HER2 status on residual disease after preoperative therapy can be helpful in selecting patients at different risk of relapse, to be included in prospective trial exploring further adjuvant therapy.

2013 - Preoperative letrozole plus lapatinib/placebo for HR+/HER2 negative operable breast cancer: biomarker analyses of the randomized phase II LET-LOB study [Poster]
Valentina, Guarneri; Antonio, Frassoldati; Daniele Giulio, Generali; Alberto, Bottini; Katia, Cagossi; Giancarlo, Bisagni; Corrado, Boni; Luigi, Cavanna; Piacentini, Federico; Bettelli, Stefania Raffaella; Guido, Ficarra; Maiorana, Antonino; Luca, Marini; Pier Franco, Conte
abstract

Background: This is a randomized, double-blind, placebo controlled study aimed to evaluate the clinical and biological effects of letrozole + lapatinib or placebo as neoadjuvant therapy in previously untreated hormone receptor positive/HER2 negative operable breast cancer. Methods: 92 postmenopausal patients with stage II-IIIA breast cancer were randomly assigned to 6 months letrozole-lapatinib (Arm A, n=43) or letrozole-placebo (Arm B, n= 49). Clinical response was evaluated according to RECIST. The following biomarkers were centrally evaluated by IHC on diagnostic core biopsy and on surgical specimens: HER2, Ki-67, EGFR, pAKT, PTEN. PIK3CA mutations were evaluated by pyrosequencing. Results: 81 patients were evaluable by USG, 8 were assessed with mammography and/or palpation. Three patients who discontinued therapy and withdrew consent were counted as non-responders according to the ITT analysis. No differences in terms of objective response rate (partial+complete response) were observed between the two arms (70% vs 63%). The percentage of patients achieving disease progression, disease stabilization, partial response and complete response were 2%, 23%, 58%, 12% respectively in the letrozole-lapatinib arm, and 6%, 29%, 61%, 2% respectively in the letrozole-placebo arm. No patients achieved pCR. All the patients were centrally confirmed as having HER2 negative disease. A significant decrease in Ki67 and pAKT expression from baseline to surgery was observed in both arms. A trend for a greater Ki67 suppression was observed in responding patients (mean Ki67 suppression -8.8 in responders vs -3.6 in non responders, p= 0.06). A mutation in PIK3CA exon 9 or 20 was observed in 37% of the patients. Overall, no differences in response were observed according to PIK3CA mutations, however, in the letrozole-lapatinib arm, the probability of achieving a clinical response was significantly higher in the PIK3CA mutation subgroup (ORR 93% vs 63% in PIK3CA WT, Pearson’s chi2 p=0.040). Conclusions: This is the first trial showing a significant correlation between PIK3CA mutation and response to letrozole-lapatinib in Hormone Receptor +/HER2- disease.

2013 - Prognostic impact of estrogen receptor (ER) level changes during progression for patients with both ER-positive (ER+) primary breast cancer and paired recurrence [Poster]
Dieci, Maria Vittoria; Barbieri, Elena; Aicha, Goubar; Andre, Fabrice; Omarini, Claudia; Bettelli, Stefania Raffaella; Piacentini, Federico; Guido, Ficarra; Pier, Franco Conte; Guarneri, Valentina
abstract

Background: We have previously reported that ER+ breast cancer (BC) patients (pts) who become ER-negative at relapse have a poorer overall survival (OS) as compared to those still ER+ at relapse [Dieci et al., Ann Oncol 2013]. Our aim is to evaluate whether, among the group of patients with an ER+ status on both primary and recurrence, changes in the level of ER expression may be of prognostic value. Methods: A total of 81 pts with ER+ primary BC and ER+ paired recurrence who underwent relapse biopsy at Modena University Hospital were studied. ER status was assessed by IHC and the cutoff for ER-positivity was >=10%. Samples were defined as ER-high (>50%) and ER-low (>=10% and <=50%). HER2-status was defined according to IHC and/or FISH results. OS was calculated as the time interval between primary BC diagnosis and death or last follow up. Results: Biopsied recurrences were: distant (86%) and local relapses (14%). Fifteen percent of primary and 21% of recurrent tumors were HER2-positive. Sixty-two pts maintained the same ER level (i.e. high or low) on both primary and relapse (ER-level concordant), whereas 19 changed from ER-high to ER-low or viceversa (ER-level discordant). No difference in OS was observed between the ER-level concordant and the ER-level discordant groups (p=0.3). However, we identified those pts whose ER-high primary BC turned into ER-low as having a particularly poor outcome. Indeed, 10yrs-OS rates were 51% for the ER-level concordant group, 50% for pts changing from ER-low to ER-high and 14% for pts changing from ER-high to ER-low (p=0.0019). Finally, we focused on the subset of pts starting from an ER-high primary BC and showing an ER+/HER2-negative phenotype on both primary and relapse (n=51). The drop of ER-level expression below the 50% cut-off at relapse was confirmed as a poor prognostic factor, as compared to pts maintaining an ER-high level (10yrs-OS 53% vs 17%, p=0.0063). Conclusions: We demonstrated that, even in the case of maintenance of the same single-receptor status (ER+) and/or tumor phenotype (ER+/HER2-negative) between primary BC and recurrence, relapse biopsy may provide relevant prognostic information.

2012 - Discordance in receptor status between primary and recurrent breast cancer has a prognostic impact: a single Institution analysis [Articolo su rivista]
Dieci, Maria Vittoria; Barbieri, Elena; Piacentini, Federico; Ficarra, G; Bettelli, Stefania Raffaella; Dominici, Massimo; Conte, Pierfranco; Guarneri, Valentina
abstract

Background: Tumor phenotype may change during breast cancer progression. This study evaluates the prognostic impact of receptor discordance between paired primaries and recurrences.Patients and Methods: 139 patients underwent histological sampling of suspected breast cancer recurrence. All the pathology assessments (ER, PgR and HER2) on both primaries and confirmed recurrences were performed at the same laboratory. Results: A breast cancer recurrence was confirmed in 119 cases. Rates of discordance were 13.4%, 39% and 11.8% for ER, PgR and HER2, respectively. Ninety-two patients maintained the same tumor phenotype (i.e., the same hormone receptors and HER2 status) whereas 27 (22.7%) changed during progression. The loss of hormone receptor-positivity and the loss of HER2-positivity resulted in a worse post-recurrence survival (p=0.01 and p=0.008, respectively) and overall survival (p=0.06 and p=0.0002, respectively), compared to the corresponding concordant-positive cases. Tumor phenotype discordance was associated with worse post-recurrence and overall survival (p=0.006 and p=0.002, respectively); those cases who turned into triple-negative experienced the poorest outcome, respect to the concordant group (p=0.001, overall survival).Conclusions: We demonstrated for the first time an impact on overall survival of phenotype discordance between primary breast cancer and relapse. Among discordant cases, receptor-loss resulted the main determinant of poorer outcome.

2012 - Predictors of human epidermal growth factor receptor 2 fluorescence in-situ hybridisation amplification in immunohistochemistry score 2+ infiltrating breast cancer: a single institution analysis. [Articolo su rivista]
Dieci, Maria Vittoria; Barbieri, Elena; Bettelli, Stefania Raffaella; Piacentini, Federico; Omarini, Claudia; Ficarra, G; Balduzzi, Sara; Dominici, Massimo; Conte, Pierfranco; Guarneri, Valentina
abstract

Eligibility for anti-human epidermal growth factor receptor 2 (HER2) treatments in breast cancer requires a correct HER2 status assessment. Testing guidelines recommend fluorescence in-situ hybridisation (FISH) for samples scored as 2+ by immunohistochemistry. This study investigates the correlation between pathological features and FISH amplification in HER2 2+ breast cancer cases.Methods: 480 HER2 2+ breast cancer samples were included. The association between tumour grade, hormone receptor status, proliferation index (Ki67) and FISH amplification, using both US Food and Drug Administration (ratio ≥2) and American Society of Clinical Oncologists/College of American Pathologists cut-offs (ratio >2.2) was evaluated.Results: 90.2% of the samples were hormone receptor positive. The median Ki67 value was 23.5%; 311 (64.8%) samples showed a Ki67 value of 15% or greater. Tumour grade was evaluable in 421 cases (87.7%), 268 (55.8%) being grade 3. FISH amplification rates were 27.5% (ratio ≥2.0) and 20.8% (ratio >2.2). Grade 3 tumours were more frequently amplified than grades 1-2 tumours: 34% versus 18% (ratio ≥2.0, p<0.001) and 27% versus 9% (ratio >2.2, p<0.001). Samples with Ki67 of 15% or greater showed higher amplification ratesthan low Ki67 samples: 31% versus 21% (ratio ≥2.0, p=0.022) and 25% versus 12% (ratio >2.2, p=0.003). The OR for FISH amplification was significant in the case of grade 3 and high Ki67 with both cut-offs.Conclusions: In this study, high tumour grade and high Ki67 significantly predicted FISH amplification in 480 HER2 2+breast cancer samples.

2012 - Prognostic role of HER2 loss after neoadjuvant therapy in patients with HER2-positive operable breast cancer [Abstract in Rivista]
Guarneri, Valentina; Barbieri, Elena; Piacentini, Federico; Dieci, Maria Vittoria; Omarini, Claudia; G., Ficarra; Bettelli, Stefania Raffaella; Conte, Pierfranco
abstract

Background: Emerging literature data are consistently showing that a change in HER2 status adversely affect the prognosis of breast cancer patients. Aim of the present analysis is to evaluate the prognostic impact of HER2 loss in breast cancer patients with HER2 positive disease treated with neoadjuvant therapy with or without anti-HER2 agents. Methods: A total of 94 HER2 positive patients were identified from a prospectively maintained database. The first cohort (A) includes 40 patients treated with chemotherapy alone (enrolled before 2005). The second cohort (B) includes 54 patients treated with neoadjuvant chemotherapy in combination with anti-HER2 agents (trastuzumab and/or lapatinib). HER2 expression was evaluated by IHC or FISH on pre-treatment core biopsy and on surgical specimen after neoadjuvant therapy. Patients were considered as having HER2 positive disease in case of IHC 3+ or FISH amplification. Results: No imbalance in terms of age, stage at diagnosis, tumor grade and expression of hormone receptor was observed in the two cohorts. In detail, 67% and 61% of the patients have a co-expression of HER2 and hormone receptor in cohort A and B, respectively. The rate of breast conservation was significantly higher in cohort B (chemotherapy+anti-HER2 agents) as compared to cohort A (chemotherapy alone) (59% vs 38%, p=0.048). Similarly, the rate of pathologic complete response (pCR) was significantly higher in cohort B (42.6% vs 7.5% in cohort A, p<0.001). A change in HER2 expression from biopsy to post-therapy samples was observed in 35% of the patients in cohort A vs 9% of the patients in cohort B (p=0.04). No patients achieving a pCR have recurred so far vs 25% of the patients with less than pCR (p=0.005). The rate of recurrence was significantly higher for patients experiencing a change in HER2 expression (47% vs 15% in patients with no change, p=0.007). At 5 years, 53% of the patients with Her2 change and 75% of the patients without Her2 change were alive and free of recurrence (log rank test: p=0.03). Conclusions: The rate of HER2 loss was significantly higher in patients not receiving anti-HER2 agents as a part of the neoadjuvant therapy. In this series, the change in HER2 status has a negative prognostic impact.

2011 - Atypical Spitzoid melanocytic tumors:A morphological, mutational, and FISH analysis [Articolo su rivista]
Massi, D.; Cesinaro, A. M.; Tomasini, C.; Paglierani, M.; Bettelli, Stefania Raffaella; Dal, Maso; Simi, L.; Salvianti, F.; Pinzani, P.; Orlando, C.; De Giorgi, V.; Lukic, S.; Maiorana, Antonino; Santucci, M.; Canzonieri, V.
abstract

Background: Identification of the clinical behavior of atypical Spitzoid tumors with conflictinghistopathologic features remains controversial.Objective: We sought to assess whether molecular findings may be helpful in the diagnostic andprognostic assessment of atypical Spitzoid tumors.Methods: A total of 38 controversial, atypical Spitzoid lesions ($1 mm in thickness) were analyzed forclinicopathological features, chromosomal alterations by fluorescence in situ hybridization (FISH) analysis(RREB1/MYB/CCND1/CEP6), BRAFV600E mutation by allele-specific real-time polymerase chain reactionconfirmed by sequencing, and H-RAS gene mutation by direct sequencing.Results: Atypical Spitzoid lesions developed in 21 female and 17 male patients (mean age 22 years). Ninepatients underwent sentinel lymph node biopsy and a sentinel lymph node micrometastasis was detectedin 4 of these 9 cases. Four additional patients, who did not receive a sentinel lymph node biopsy,experienced bulky lymph node metastases and one experienced visceral metastases and death. Lesionsfrom patients with lymph node involvement showed more deep mitoses (P \ .01), less inflammation(P = .05), and more plasma cells (P = .04). FISH analysis demonstrated the presence of chromosomalalterations in 6 of 25 cases. Correlation with follow-up data showed that the only case with fatal outcomeshowed multiple chromosomal alterations by FISH analysis. BRAFV600E mutation was detected in 12 of 16cases (75%) and H-RAS mutation on exon 3 was found in 3 of 11 cases (27%).Limitations: Our results require validation in a larger series with longer follow-up information.Conclusions: FISH assay may be of help in the prognostic evaluation of atypical Spitzoid tumors.Diagnostic significance of BRAFV600E and H-RAS mutations in this setting remains unclear. ( J Am AcadDermatol 2011;64:919-35.)

2011 - Change in HER2 Status in HER2 Positive Operable Breast Cancer Patients Treated with Neoadjuvant Chemotherapy with or without Anti-HER2 Therapy: Analysis of Two Consecutive Cohorts [Abstract in Rivista]
Barbieri, Elena; Piacentini, Federico; Dieci, Maria Vittoria; Ficarra, G; Bettelli, Stefania Raffaella; Conte, Pierfranco; Guarneri, Valentina
abstract

Introduction: emerging literature data have shown a change of HER2 expression from primary tumors to metastatic deposits. Tumor heterogeneity, genetic drift as well as selective pressure of adjuvant therapy have been suggested to explain this phenomenon. Aim of the present analysis is to evaluate the change in HER2 expression after neoadjuvant chemotherapy with or without anti-HER2 agents. Methods: two consecutive cohorts of HER2+ breast cancer patients treated with neoadjuvant therapy were identified from a prospectively maintained database including 310 patients. The first cohort (A) includes 38 patients enrolled before 2005, treated with chemotherapy alone. The second cohort (B) includes 48 patients treated with neoadjuvant chemotherapy in combination with antiHER2 agents (trastuzumab or lapatinib). HER2 expression was evaluated by IHC on pre-treatment core biopsy (tru-cut with 14 gauge needle) and on surgical specimen after neoadjuvant therapy. FISH analysis was performed on IHC 2+ samples. Results: The two cohorts were balanced in respect of tumor stage, patient age, and HR expression. In particular, a co-expression of HER2 and HR was observed in 60% of the patients in cohort A and in 70% of the patients in cohort B (p=0.2). Patients in cohort B have a significantly higher rate of pathologic complete response (pCR) in comparison to cohort A (45% vs 11%, p=0.001). A change in HER2 expression from biopsy to post-therapy samples was observed in 39% of the patients in cohort A vs 12% of the patients in cohort B (p=0.02). No patients with pCR have recurred so far vs 25% of the patients with less than pCR (p=0.005). The rate of recurrence was significantly higher for patients experiencing a change in HER2 expression (50% vs 19%, p=0.018). Conclusion: contrary to our expectations, patients not receiving anti-HER2 therapy as part of neoadjuvant therapy were more likely to have a change in HER2 status vs patients receiving anti-HER2 neoadjuvant therapy. The change in HER2 status has a negative prognostic impact.

2011 - Correlation between CD133 expression and mgmt status in recurrences melanoma [Abstract in Rivista]
Migaldi, Mario; Reggiani Bonetti, L.; Cesinaro, A. M.; Maiorana, Antonino; Farinetti, Alberto; Bettelli, S.; Sgambato, A.
abstract

...

2011 - Cytomegalovirus infection of the upper gastrointestinal tract: a clinical and pathological study of 30 cases. [Articolo su rivista]
Reggiani Bonetti, L.; Losi, L.; Di Gregorio, C.; Bertani, Angela; Merighi, A.; Bettelli, S.; Scuri, M.; Maiorana, Antonino
abstract

OBJECTIVE:The study reviews the endoscopic and histological features of human cytomegalovirus (HCMV) infections of the upper gastrointestinal (UGI) tract.MATERIALS AND METHODS:Clinical histories, endoscopic findings and bioptic specimens of 30 cases of HCMV infection of the UGI tract, diagnosed in a University Hospital in a 10-year period, were reviewed. In all cases, viral inclusion bodies were detected in routine histopathological sections and the diagnosis was confirmed with immunohistochemistry.RESULTS:Six patients were HIV+, whereas four had received organ transplantations, one was affected by common variable immunodeficiency and four had a recent history of malignancy. No other pathologic condition was evidenced in the remaining 15 cases. Mucosal alterations were endoscopically observed in the stomach (19 cases), esophagus (9), cardias (6) and duodenum (1), and multiple organs being synchronously affected in five patients (3 HIV+, 2 with history of malignancy). The antropyloric area was the most frequently affected site. Single ulcers were detected in 11 cases and multiple ulcers in 8, whereas mucosal thickenings (in the form of localized thickenings, polyps or rugal hypertrophy) were present in 13 patients. Thickenings of the mucosa were detected only in the stomach. At histology, necrotic material and granulation tissue were associated with moderate or marked lympho-plasmacytic infiltrate and foveolar hyperplasia in ulcerative lesions, whereas lesions labeled as mucosal thickenings showed mild or moderate chronic inflammatory infiltrate and foveolar hyperplasia.CONCLUSIONS:Endoscopic manifestations of UGI tract involvement in HCMV infection are not specific, varying from erythematous mucosa to ulcers to mucosal thickenings.

2011 - Morphological analysis of three extrathoracic bronchogenic cysts simulating neoplasms [Articolo su rivista]
Lupi, M.; Reggiani Bonetti, L.; Stauder, E.; Bettelli, S.; Maiorana, A.
abstract

Bronchogenic cyst is a congenital anomaly of the primitive foregut. Unusual occurrences in extrathoracic sites have been described in the literature, some of which may clinically simulate neoplasms. We report three additional cases arising near the left adrenal gland, left ovary and gastric wall. Pathological findings are discussed, together with a review of the literature.

2011 - Trans-CHER-Lob: A Biomarker Analysis of the Randomized Phase II Study of Neoadjuvant Chemotherapy Plus Trastuzumab, Lapatinib or Combined Trastuzumab and Lapatinib in HER2 Positive Operable Breast Cancer. [Abstract in Rivista]
Guarneri, Valentina; Frassoldati, A; Ficarra, G; Maiorana, Antonino; Bettelli, Stefania Raffaella; Bottini, A; Cagossi, K; Bisagni, G; Ravaioli, A; Amadori, D; Musolino, A; Cavanna, L; Orlando, L; Giardina, G; Piacentini, Federico; Bagnalasta, M; Conte, Pierfranco
abstract

Introduction: The CHER-Lob study is a randomized phase II trial of preoperative sequential taxanes-anthracyclines in combination with trastuzumab, lapatinib, or combined trastuzumab and lapatinib in HER2 positive, stage II-IIIA breast cancer patients. A translational program to evaluate predictors of response as well as treatment effects on tissue biomarkers was pre-planned. Methods: The CHER-Lob translational program includes the central evaluation of HER2, p95HER2, PTEN, pAKT, Ki67 on pre and post-therapy samples. All these biomarkers have been evaluated by immunohistochemistry. FISH analysis was performed in case of HER2 IHC 2+, and in all the discordant cases between central and local laboratories. Biomarkers change from baseline to surgery has been evaluated with the Wilcoxon signed-ranks matched-pair test. Results: 121 patients have been randomized. The pathologic complete response rate (breast and axillary lymphnodes) was 26% in Arm A (chemotherapy + trastuzumab), 28% in arm B (chemotherapy + lapatinib) and 44% in arm C (chemotherapy + trastuzumab and lapatinib). The concordance between central and local HER2 assessment on pre-treatment biopsy was 97%. The mean (min;max) PTEN expression pre- and post-therapy were 66% (0;100) and 68.4% (0;100) respectively. The mean (min;max) pAKT expression pre- and post-therapy were 23.3% (0;100) and 8.8% (0;90) respectively. A significant decrease was observed in the overall samples (p=0.01). When analyzing the lapatinib alone arm, the difference was no longer significant (p=0.06), while it maintained significance when evaluating the two trastuzumab containing arms (p=0.0013). The mean (min;max) ki67 expression pre- and post-therapy were 29.5% (4;90) and 16.6% (1;50) respectively. A significant decrease was observed when looking at the whole population (p<0.0001). A significantly higher ki67 inhibition was observed in the dual vs single anti-Her2 therapy (p= 0.003). Conclusions: The central HER2 retesting showed a high concordance with local laboratories. Treatment induced a suppression in pAKT expression, that was higher in patients receiving trastuzumab. The dual anti-HER2 blockade induced a higher KI67 inhibition as compared to single anti-Her2 blockade. The evaluation of the predictive and prognostic role of these biomarkers is ongoing. Supported by GlaxoSmithKline

2010 - 35. PREDICTORS OF HER2 FISH POSITIVITY IN PRIMARY BREAST CANCER DIAGNOSIS SCORED 2+ WITH IHC: RESULTS OF A SINGLE INSTITUTION ANALYSIS [Abstract in Atti di Convegno]
Barbieri, E.; Bettelli, S.; Piacentini, F.; Ficarra, G.; Conte, Pf.; Guarneri, V.
abstract

Background: HER-2 positivity in breast cancer is defined as a 3+ score in immunohistochemistry (IHC) assay (defined as uniform intense membrane staining of > 30% of invasive tumor cells) or FISH amplification (ratio of HER-2 to CEP17 of > 2.2 or average HER-2 gene copy number > six signals/nucleus for those test systems without an internal control probe). In case of 2+ staining at IHC, the test is considered equivocal, and FISH is mandatory to verify the HER-2 status. Up to 15% of breast cancer are IHC 2+. Aim of this study was to identify potential predictors of FISH positivity for those cases scored IHC 2+. Methods: During 2009, a total of 200 samples were processed for FISH assay. Among them, 161 (80.5%) were primary breast diagnoses with HER2 IHC score2+. Twelve cases (7.5%) resulted not evaluable, leaving 149 cases eligible for this analysis. We have evaluated the correlation between FISH positivity and hormone receptor expression, nuclear grade and proliferation. Results: Mean age of the patients was 64 years (range 25-97). Hormone receptors status was known in 125/149 patients (84%); 107 (86%) were ER +, 92 (74%) were PgR + , 110 (88%) were either ER or PgR +. MIB-1 was available in 121 cases (81%). The mean Ki67 value was 20% (range: 2 to 80). Nuclear grade was available in 108 cases (72.5%), 50 cases were G1 or G2 (46%) and 58 were G3 (54%). FISH resulted amplified in 41 pts. (27.5%). None of the evaluated factors was significantly associated with the probability of FISH positivity: the odds ratios for FISH positivity were 1.005 for age <50 vs >50 yrs. (p= 0.704), 1.001 for proliferation (p=0.927), 1.497 for grade 3 vs. 1-2 (p=0.269), 0.96 for hormone receptor negative vs. positive (0.960). Conclusions: In this study, hormone receptor status, nuclear grade and proliferation were not significantly associated with the probability of FISH positivity. The percentage of hormone receptor positivity in this population was surprisingly high, and we are evaluating the prevalence of hormone receptor positivity in patients with HER2 IHC 0/1+ and 3+ diagnosed during the same year . The study is ongoing to include cases diagnosed between 2000-2008

2010 - Alterations of 9p21 analysed by FISH and MLPA distinguish atypical spitzoid melanocytic tumours from conventional Spitz's nevi but do not predict their biological behaviour. [Articolo su rivista]
Cesinaro, Am; Schirosi, L; Bettelli, S; Migaldi, Mario; Maiorana, Antonino
abstract

Alterations of 9p21 analysed by FISH and MLPA distinguish atypical spitzoid melanocytic tumours from conventional Spitz's nevi but do not predict their biological behaviour Aim:  Histopathological criteria alone cannot predict the biological behaviour of spitzoid melanocytic tumours. The aim of this study was to investigate whether 9p21 status influence the prognosis of the spitzoid melanocytic tumours, peculiar lesions whose biological behaviour cannot be predicted by histopathological criteria alone. Methods and results:  Twenty-eight atypical spitzoid tumours, 12 conventional Spitz's nevi and one congenital Spitz's nevus were studied by fluorescent in-situ hybridization (FISH) and multiple ligation-dependent probe amplification (MLPA) for the presence of 9p21 deletion. The 28 patients were aged 3-56 years (mean 32, median 35), and follow-up ranged between 4 and 156 months (mean 51, median 48). Eight patients (28.5%) experienced lymph node metastasis (three cases with macrometastasis and five with micrometastasis). Of those with macrometastasis, two are alive after 159 and 26 months, whereas a third developed widespread metastases and died after 26 months. All of the other patients are alive. Statistically, the thickness (P = 0.01) and the diameter (P = 0.009) of the lesions significantly correlated with metastasis. Deletion of 9p21 by FISH analysis was observed in eight spitzoid tumours (28.5%), and MLPA demonstrated alterations of 9p21, particularly deletion of CDKN2A, in the same lesions, whereas all Spitz's nevi, except the congenital one, were of unaltered 9p21 status (P < 0.0001). Deletion of 9p21/CDKN2A did not correlate with the presence of metastasis. Conclusion:  Alterations at 9p21 locus are significantly more frequent in spitzoid tumours than in Spitz's nevi, but do not predict their biological behaviour.

2010 - Cerebrospinal fluid cytology in a case of primary diffuse leptomeningeal and pineal melanocytic lesion, with histological confirmation [Articolo su rivista]
Zunarelli, Elena; Bettelli, Stefania; Reggiani-Bonetti, Luca; Bertolini, Federica; Falasca, Angelo
abstract

Non available

2010 - EGFR polysomy in squamous cell carcinoma of the thyroid. Report of two cases and review of the literature [Articolo su rivista]
Bonetti, L. R.; M., Lupi; M., Trani; N., Trani; Sartori, Giuliana; Schirosi, Laura; Bettelli, Stefania Raffaella; G., Zanelli; L., Maccio; Maiorana, Antonino
abstract

AIMS AND BACKGROUND:Primary squamous cell carcinoma of the thyroid gland (PSCCT) is an uncommon malignancy characterized by a poor prognosis. A radical surgical approach combined with radiotherapy or chemotherapy is the generally accepted treatment for this tumor. The epidermal growth factor receptor (EGFR) is a transmembrane tyrosine kinase receptor modulating the cell proliferation and biological progression of many human epithelial tumors. The EGFR overexpression in PSCCT suggests an additional therapeutic option for the treatment of this tumor.METHODS AND STUDY DESIGN:The clinicopathological features and immunohistochemical profiles of two cases of primary squamous cell carcinoma of the thyroid in a 66-year-old and an 83-year-old woman are presented. EGFR status was valued in both cases.RESULTS:Overexpression of EGFR protein was detected in 50% and 75% of the tumor cell membranes. EGRF gene polysomy was detected in both tumors.CONCLUSIONS:Pharmaceuticals targeting EGFR may help to provide the rationale for an additional, novel therapeutic option for this rare tumor, especially when other therapeutic options have been exhausted.

2010 - Prognostic role of EGFR gene copy number and KRAS mutation in patients with locally advanced rectal cancer treated with preoperative chemoradiotherapy. [Articolo su rivista]
C., Bengala; Bettelli, Stefania Raffaella; F., Bertolini; G., Sartori; Fontana, Annalisa; N., Malavasi; R., Depenni; S., Zironi; DEL GIOVANE, Cinzia; G., Luppi; Conte, Pierfranco
abstract

Epidermal growth factor receptor (EGFR), evaluated by immunohistochemistry, has been shown to have prognostic significance in patients with colorectal cancer. Gene copy number (GCN) of EGFR and KRAS status predict response and outcome in patients treated with anti-EGFR therapy, but their prognostic significance in colorectal cancer patients is still unclear.We have retrospectively reviewed the baseline EGFR GCN, KRAS status and clinical outcome of 146 locally advanced rectal cancer (LARC) patients treated with preoperative chemoradiotherapy. Pathological response evaluated by Dworak's tumour regression grade (TRG), disease-free survival (DFS) and overall survival (OS) were analysed.Tumour regression grade 4 and TRG3-4 were achieved in 14.4 and 30.8\% of the patients respectively. Twenty-nine (19.9\%) and 33 patients (19.2\%) had an EGFR/nuclei ratio &gt;2.9 and CEP7 polisomy &gt;50\% respectively; 28 patients (19.2\%) had a KRAS mutation. Neither EGFR GCN nor KRAS status was statistically correlated to TRG. 5-year DFS and OS were 63.3 and 71.5\%, respectively, and no significant relation with EGFR GCN or KRAS status was found.Our data show that EGFR GCN and KRAS status are not prognostic factors in LARC treated with preoperative chemoradiation.

2009 - Epidermal growth factor receptor gene copy number, K-ras mutation and pathological response to preoperative cetuximab, 5-FU and radiation therapy in locally advanced rectal cancer.. [Articolo su rivista]
Bengala, C; Bettelli, Stefania Raffaella; Salvi, S; Chiara, S; Sonaglio, C; Losi, Lorena; Bigiani, N; Sartori, G; Dealis, Cristina; Malavasi, Norma; D'Amico, Roberto; Luppi, G; Gatteschi, B; Maiorana, Antonino; Conte, Pierfranco
abstract

Cetuximab improves activity of chemotherapy in metastatic colorectal cancer (mCRC). Gene copy number (GCN) of epidermal growth factor receptor (EGFR) has been suggested to be a predictive factor of response to cetuximab in patients (pts) with mCRC; on the contrary, K-ras mutation has been associated with cetuximab resistance. PATIENTS AND METHODS: We have conducted a phase II study with cetuximab administered weekly for 3 weeks as single agent and then with 5-fluorouracil and radiation therapy as neo-adjuvant treatment for locally advanced rectal cancer (LARC). EGFR immunohistochemistry expression, EGFR GCN and K-ras mutation were evaluated on diagnostic tumor biopsy. Dworak's tumor regression grade (TRG) was evaluated on surgical specimens. RESULTS: Forty pts have been treated; 39 pts are assessable. TRG 3 and 4 were achieved in nine (23.1%) and three pts (7.7%) respectively; TRG 3-4 rate was 55% and 5.3% in case of high and low GCN, respectively (P 0.0016). Pts with K-ras mutated tumors had lower rate of high TRG: 11% versus 36.7% (P 0.12). In pts with wild-type K-ras, TRG 3-4 rate was 58.8% versus 7.7% in case of high or low GCN, respectively (P 0.0012). CONCLUSIONS: In pts with LARC, EGFR GCN is predictive of high TRG to cetuximab plus 5-FU radiotherapy. Moreover, our data suggest that a wild-type K-ras associated with a high EGFR GCN can predict sensitivity to cetuximab-based treatment.PMID: 19095777 [PubMed - indexed for MEDLINE]Related articlesNeoadjuvant treatment with single-agent cetuximab followed by 5-FU, cetuximab, and pelvic radiotherapy: a phase II study in locally advanced rectal cancer. Int J Radiat Oncol Biol Phys. 2009 Feb 1; 73(2):466-72. Epub 2008 Nov 10. [Int J Radiat Oncol Biol Phys. 2009]Expression of epiregulin and amphiregulin and K-ras mutation status predict disease control in metastatic colorectal cancer patients treated with cetuximab. J Clin Oncol. 2007 Aug 1; 25(22):3230-7. [J Clin Oncol. 2007]A polymorphism of EGFR extracellular domain is associated with progression free-survival in metastatic colorectal cancer patients receiving cetuximab-based treatment. BMC Cancer. 2008 Jun 10; 8:169. Epub 2008 Jun 10. [BMC Cancer. 2008]Review[The RAS paradox of the EGFR-targeted therapy in colorectal cancer] Magy Onkol. 2008 Jun; 52(2):185-91. [Magy Onkol. 2008]ReviewAssessment of somatic k-RAS mutations as a mechanism associated with resistance to EGFR-targeted agents: a systematic review and meta-analysis of studies in advanced non-small-cell lung cancer and metastatic colorectal cancer. Lancet Oncol. 2008 Oct; 9(10):962-72. Epub 2008 Sep 17. [Lancet Oncol. 2008]» See reviews... | » See all... Patient Drug InformationFluorouracil (Adrucil®) Your doctor has ordered the drug fluorouracil to help treat your illness. The drug is given by injection into a vein. Source: AHFS Consumer Medication Information

2009 - Expression of estrogen receptor in hemangioma of the uterine cervix: Reports of three cases and review of the literature [Articolo su rivista]
Reggiani Bonetti, Luca; Boselli, F.; Lupi, M.; Bettelli, S.; Schirosi, L.; Bigiani, N.; Sartori, G.; Rivasi, Francesco
abstract

The occurrence of hemangioma in the female genital tract, particularly in uterine cervix, is rare. The majority of them show asymptomatic behavior. Surgical excision remains curative in most of the cases. Conservative therapies such as sclerosing agents, cryotherapy, and CO(2) laser excision may be alternatively applied. We present three cases of hemangiomas of the cervix in asymptomatic women, diagnosed as cavernous hemangioma in two cases and capillary hemangioma in one. All tumors were immunoreactive for CD31, CD34, factor-VIII-related antigen. Focal expression of estrogen receptors was detected. No positivity was obtained with progesterone receptor antibodies. The presence of estrogen receptor in the endothelial cells of the hemangioma of the cervix suggests a direct role of this hormone in the hemangioma development. A possible target therapy is discussed.

2008 - Alveolar rhabdomyosarcoma of the uterine cervix. A case report confirmed by FKHR break-apart rearrangement using a fluorescence in situ hybridization probe on paraffin-embedded tissues. [Articolo su rivista]
Rivasi, Francesco; Botticelli, L; Bettelli, Sr; Masellis, G.
abstract

Alveolar rhabdomyosarcoma (ARMS) is a very rare malignant soft tissue tumor found in the uterine cervix. In fact, to our knowledge, only 3 cases have previously been described in the literature. A 49-year-old premenopausal woman who had a history of vaginal bleeding underwent hysterectomy for multiple uterine nodules. Pathologic examination showed multiple leiomyomas in the uterus and ARMS in the cervix. An FKHR break-apart rearrangement by fluorescence in situ hybridization (FISH) probe on paraffin-embedded tissues identified specific chromosomal abnormalities. Systemic chemotherapy and pelvic irradiation were also performed. She remains in clinical remission from neoplastic recurrences 14 months after surgery. The literature has been reviewed; the histologic differential diagnosis of this rare lesion has been discussed and FISH has been suggested to be useful in differentiating ARMS from other poorly differentiated round cell tumors.

2008 - COMPARISON OF HER-2 STATUS IN PRIMARY BREAST CANCER AND METASTATIC SITES: RESULTS FROM A SINGLE INSTITUTION ANALYSIS [Relazione in Atti di Convegno]
Guarneri, V.; Giovannelli, S.; Ficarra, G.; Bettelli, S.; Piacentini, F.; Barbieri, E.; Dieci, Mv.; Jovic, G.; Conte, Pf.
abstract

Introduction:HER-2 overexpression occurs in 15-20% of breast cancer; the assessment of HER2 status is necessary to select patients who are candidate to receive anti-HER2 agents, such as trastuzumab. The evaluation of HER2 is generally carried out in primary tumor. However, it is controversial if it would be more appropriate to reassess HER2 status in the metastatic lesion. Aim of the present analysis is to compare the HER2 status in primary tumors versus metastatic sites in metastatic breast cancer patients. Patients and Methods: The Archive of the Pathology Division was searched to identify all the biopsies performed since 2004 resulted positive for metastases from breast cancer. Among these patients, only those with available samples from primary tumor and followed at our department were considered for this analysis. Therefore, 68 out of the previously 89 identified patients are included in the present study. HER-2 overexpression was evaluated by IHC and/or by FISH. Overall, samples were considered positive for HER2 in case of IHC 3+, or 2+ and FISH amplification, or FISH amplification. In case of discordance between IHC and FISH, HER 2 status was defined according to the FISH result. Results: Median age at diagnosis was 53 years (range 27-74). Among the 49 patients evaluable by IHC, 40 (82%) were negative (0-1+); 3 (6%) were IHC 2+, and 6 (12%) were 3+. Among the 38 patients evaluable by FISH, 32 (84%) were non-amplified, and 6 (16%) were amplified. Overall, at diagnosis, 15% of tumors were HER2 positive. Sites of biopsied metastases were: soft tissues (n=22), liver (n=20), chest wall (n=13), CNS (n=4), bronchus (n=4), bone (n=1), bone marrow (n=2), and other (n=2). On paired metastases, HER2 status was found to be unchanged in 57 out of the 68 (84%) evaluable patients, while a change in HER2 status was observed in 11 cases (16%). All the discordant cases were assessed both by IHC and FISH whenever possible, or by using the same modality. Surprisingly, only 2 patients changed from HER2 positivity to HER2 negativity (without receiving anti HER2 therapy prior to metastatic site biopsies), while in 9 patients her2 was negative on primary tumors and overexpressed in paired metastases. Conclusion. The unexpected discordance observed in our analysis, with particular regard to those tumors that convert from negative to positive HER2 status, warrants further studies with larger series. However, based on these data, when feasible with minimal invasiveness, a biopsy of metastatic disease might increase treatment options for a significant proportion of patients.

2008 - Comparison of HER-2 and hormone receptor expression in primary breast cancers and asynchronous paired metastases: impact on patient management [Articolo su rivista]
Guarneri, Valentina; Giovannelli, Simona; Ficarra, G; Bettelli, Stefania Raffaella; Maiorana, Antonino; Piacentini, Federico; Barbieri, Elena; Dieci, Maria Vittoria; D'Amico, Roberto; Jovic, Gordana; Conte, Pierfranco
abstract

The assessment of hormone receptors (HRs) and human epidermal growth factor receptor (HER)-2 is necessary to select patients who are candidates for hormonal and anti-HER-2 therapy. The evaluation of these parameters is generally carried out in primary tumors and it is not clear if reassessment in metastatic lesions might have an impact on patient management. The primary aim of this analysis was to compare HER-2 and HR status in primary tumors versus metastatic sites in breast cancer patients. PATIENTS AND METHODS: Seventy-five patients with available samples from primary tumors and paired metastases were included. HER-2 status was evaluated by immunohistochemistry (IHC) and/or fluorescence in situ hybridization (FISH); HR status was assessed by IHC. RESULTS: Nineteen percent of primary tumors were HER-2 positive; 77% were HR positive. Sites of biopsied or resected metastases were: locoregional soft tissues (n = 30), liver (n = 20), central nervous system (n = 5), bone (n = 5), pleura (n = 4), distant soft tissues (n = 3), abdomen (stomach, colon, peritoneum) (n = 3), bronchus (n = 3), and bone marrow (n = 2). For paired metastases, the HER-2 status was unchanged in 84% of cases; two patients changed from positive to negative, while 10 patients converted from negative to positive (agreement, 84%; kappa = 0.5681). A change in HR status was observed in 16 cases (21%): nine cases from positive to negative and seven cases from negative to positive (agreement, 78.7%; kappa = 0.4158). CONCLUSIONS: Further studies are necessary to better define the level of discordance in HER-2 or HR status between primary tumors and paired metastases. However, a biopsy of metastatic disease can be recommended, if feasible with minimal invasiveness, because treatment options might change for a significant proportion of patients

2008 - FISH analysis in cell touch preparations and cytological specimens from formalin-fixed fetal autopsies. [Articolo su rivista]
Rivasi, Francesco; Schirosi, L; Bettelli, S; Bigiani, N; Curatola, C.
abstract

Postmortem studies on still-borns and miscarriages are important to define the sex and eventually the morphologic anomalies correlated to chromosomal aberrations. When the conditions for carrying out a cytogenetic study do not exist, these chromosomal alterations can be investigated by nucleic acid fluorescent in situ hybridization (FISH), which can be performed on interphase nuclei, usually on formalin-fixed paraffin embedded tissues or on fresh cytological specimens. The objective of the present study is to prove whether this technique can be successfully applied to formalin-fixed cell touch preparations and cytological specimens obtained from foetal autopsies. The study was carried out 12 abortions some of which were spontaneous and some of which were therapeutic. The materials were formalin-fixed. Cell touch preparations and cytological specimens were obtained. The FISH was performed using X/Y probes (Vysis) and the Aneuvysion Kit (05J38-030, Vysis), the probes being for chromosomes 13/21 and X/Y/18. To verify the reliability of the technique, the same reactions were also performed on fresh analogous materials. The slides were evaluable, and the probes hybridized to interphase nuclei showed distinct signals. All the samples were adequate for FISH analysis without any notable difference in the results. Moreover, it is technically possible to perform this analysis not only on fresh but particularly on formalin-fixed cytological specimens. On the other hand, the use of this type of cytological samples, as compared to formalin-fixed and paraffin embedded tissue sections, has the advantage of presenting intact, noncut nuclei with preserved cytomorphology, avoiding the problems of overlapping nuclei and making the identification of the real chromosomal arrangement easier.

2007 - Microsatellite and EGFR, HER2 and K-RAS analyses in sclerosing hemangioma of the lung [Articolo su rivista]
Sartori, G; Bettelli, Stefania Raffaella; Schirosi, L; Bigiani, N; Maiorana, Antonino; Cavazza, Alessia; Rossi, G.
abstract

Sclerosing hemangioma (SH) is an uncommonpulmonary tumor thought to derive from primitive respiratoryepithelium consisting of 2 cell populations (cuboidal surface andpolygonal stromal cells) and sharing some clinical characteristics(frequent occurrence in nonsmoking women of Asian ethnicity)with bronchioloalveolar carcinoma with which it has beensuggested a possible common origin. We investigated 11 cases ofSH by immunohistochemistry, fluorescence in situ hybridization,and polymerase chain reaction-based microsatellite andmutational analyses with particular emphasis on possiblealterations of microsatellite loci located at tumor suppressorgenes (FHIT, p16, Rb, and p53) involved in lung adenocarcinomagenesis and EGFR, HER2, and K-RAS genes. AlthoughEGFR expression was observed in all tested cases, none showedHER2 immunostaining. Fluorescence in situ hybridization andmutational analysis of EGFR and HER2 and also K-RASsequencing did not reveal molecular alterations, whereas alleliclosses at p16 and Rb loci (4 and 2 out of 9 tested cases,respectively) with an identical microsatellite allelic loss patternin both cuboidal and polygonal cells were observed. The findingof microsatellite alterations in chromosomal regions related togenes deeply involved in early stage lung adenocarcinoma couldsuggest a possible link between SH and bronchioloalveolarcarcinoma, but tumor pathway promoted by EGFR, HER2, andK-RAS does not represent a common molecular mechanism oftumorigenesis. Microsatellite alterations identified in cuboidaland polygonal cells further confirm the clonal and neoplasticnature of both components of SH.

2005 - Does HPV play a role in the etiopathogenesis of ameloblastoma? An immunohistochemical, in situ hybridization and polymerase chain reaction study of 18 cases using laser capture microdissection [Articolo su rivista]
Migaldi, Mario; Pecorari, M.; Rossi, G.; Maiorana, Antonino; Bettelli, S.; Tamassia, Mg; DE GAETANI, Carmela; Leocata, P.; Portolani, Marinella
abstract

Ameloblastomas are epithelial tumors of odontogenic origin, biologically characterized by local recurrence. Among different etiologic factors, HPV infection has been recently postulated to be somehow involved in ameloblastoma etiopathogenesis. To address this issue, we studied 18 ameloblastomas by means of immunohistochemistry, in situ hybridization (conventional and amplified), polymerase chain reaction and nested-polymerase chain reaction analyses using laser capture microdissection in order to detect the occurrence of HPV in this setting. No evidence of HPV infection was detected by morphological examination, immunohistochemistry, in situ hybridization and conventional polymerase chain reaction, while nested-polymerase chain reaction showed a weak positive band in two cases. However, the subsequent restriction enzyme analysis carried out from the nested-polymerase chain reaction amplification products of these two samples excluded the presence of HPV subtypes 16, 18, 31, 33, 35, 52, and 58. The search for HPV 6 and 11 in the same specimens was also negative. In conclusion, our data do not support an etiopathogenetic evidence for HPV in ameloblastoma.

2004 - The prognostic role of c-kit protein expression in resected large cell neuroendocrine carcinoma of the lung. [Articolo su rivista]
C., Casali; Stefani, Alessandro; G., Rossi; Migaldi, Mario; S., Bettelli; A., Parise; Morandi, Uliano
abstract

Large cell neuroendocrine carcinoma (LCNEC) is a high-grade neuroendocrine tumor of the lung that shares some clinicopathologic and molecular features with small cell lung carcinoma (SCLC). Optimal treatment has not yet been standardized and significant prognostic factors are lacking. Because c-kit protein overexpression has been recently reported as a negative prognostic factor in SCLC we investigated its expression and prognostic value in a series of LCNEC.Resected LCNEC fulfilling the morphologic criteria of the 1999 World Health Organization classification of lung tumors and showing neuroendocrine differentiation by appropriate immunohistochemical markers were retrospectively reviewed. Immunostaining for c-kit protein expression was performed using the polyclonal antibody CD117. Clinical and pathologic characteristic were reported and analyzed and a survival study was performed.Thirty-three patients underwent radical resection. Thirty-one were male (94\%) and 32 were smokers (97\%). Ten (30.3\%), 11 (33.3\%), 5 (15.2\%), and 7 (21.2\%) were at stage IA, IB, IIB, and IIIA respectively. Overall 1-, 3-, and 5-year survival rates were respectively 79\%, 58\%, and 51\%. Survival analysis showed no differences for any of the clinicopathological features except for CD117 immunostaining: 1-year and 3-year survival rates were respectively 91\% and 82\% for CD117-negative LCNEC, and 72\% and 44\% for CD117-positive ones (p = 0.046). Positivity of CD117 was significantly related to recurrence rate: 60\% versus 23\% for CD117 positive and negative LCNEC respectively (p = 0.037).Radical resection of large cell neuroendocrine carcinoma achieves poor outcomes. The c-kit protein is frequently expressed in this neoplasia and its expression represents a negative prognostic factor. This immunohistochemical marker may represent the basic rationale to select LCNEC for novel targeted therapy.

1999 - Detection of human papillomavirus DNA in urinary bladder carcinoma by in situ hybridization [Articolo su rivista]
DE GAETANI, Carmela; G., Ferrari; E., Righi; S., Bettelli; Migaldi, Mario; P., Ferrari; Trentini, Giampaolo
abstract

To investigate the sensitivity of an in situ hybridisation system to detect human papillomavirus (HPV) infection in transitional cell bladder cancer and to evaluate the advantages of analysing multiple biopsies; to examine the correlation between HPV tumour infection detected by in situ hybridisation and the presence of serum anti-HPV antibodies detected by enzyme linked immunosorbent assay (ELISA); and to relate the presence of viral infection to grade, stage, and follow up in cases of bladder cancer. METHODS: The in situ hybridisation technique was used with broad spectrum and type specific (6/11, 16/18, 31/33/35) probes against HPV DNA in formalin fixed, paraffin embedded tissues from 43 cases of bladder cancer. The results were analysed for the presence and type of papillomavirus and correlated with clinicopathological variables. RESULTS: The presence of HPV DNA was identified by the in situ hybridisation technique in 17 of 43 cases of bladder cancer; 12 of these were serum antibody positive and 10 had had multiple biopsies. Fifteen of the cases that were negative for HPV DNA by in situ hybridisation had positive serum serology when tested by ELISA. In 14 cases, the HPV was either types 16/18 or types 31/33/35, both of which carry high oncogenic risk. The stage (p < 0.05) and grade (NS) of the tumour and the outcome on follow up (p < 0.05) were correlated with the presence of HPV infection. CONCLUSIONS: ELISA is not useful in identifying patients with HPV positive bladder cancer, but the use of several probes and multiple biopsies increases the detection rate of HPV in neoplastic tissues. The association between tumour virus infection and high grade/high stage tumours and worse outcome suggests that HPV infection of neoplastic tissue has a negative effect on the behaviour and evolution of transitional cell bladder carcinoma.