Plasma miRNA-based signatures in CRC screening programs.

Colorectal cancer (CRC) screening programs help diagnose cancer precursors and early cancers and help reduce CRC mortality. However, currently recommended tests, the fecal immunochemical test (FIT) and colonoscopy, have low uptake. There is therefore a pressing need for screening strategies that are minimally invasive and consequently more acceptable to patients, most likely blood based, to increase early CRC identification. MicroRNAs (miRNAs) released from cancer cells are detectable in plasma in a remarkably stable form, making them ideal cancer biomarkers. Using plasma samples from FIT-positive (FIT+) subjects in an Italian CRC screening program, we aimed to identify plasma circulating miRNAs that detect early CRC. miRNAs were initially investigated by quantitative real-time PCR in plasma from 60 FIT+ subjects undergoing colonoscopy at Fondazione IRCCS Istituto Nazionale dei Tumori, then tested on an internal validation cohort (IVC, 201 cases) and finally in a large multicenter prospective series (external validation cohort [EVC], 1121 cases). For each endoscopic lesion (low-grade adenoma [LgA], high-grade adenoma [HgA], cancer lesion [CL]), specific signatures were identified in the IVC and confirmed on the EVC. A two-miRNA-based signature for CL and six-miRNA signatures for LgA and HgA were selected. In a multivariate analysis including sex and age at blood collection, the areas under the receiver operating characteristic curve (95% confidence interval) of the signatures were 0.644 (0.607-0.682), 0.670 (0.626-0.714) and 0.682 (0.580-0.785) for LgA, HgA and CL, respectively. A miRNA-based test could be introduced into the FIT+ workflow of CRC screening programs so as to schedule colonoscopies only for subjects likely to benefit most.

The Effects of miR-20a on p21: Two Mechanisms Blocking Growth Arrest in TGF-ß-Responsive Colon Carcinoma.

Loss of response to TGF-ß is a central event in the genesis of colorectal cancer (CRC), a disease that, in the majority cases, is refractory to growth inhibition induced by this cytokine. However, inactivating mutations at receptors and transducers from the TGF-ß cascade occur only in approximately half of CRCs, suggesting the involvement of additional mechanisms altering the response to the cytokine. We have recently described the amplification of the 13q31 locus, where the miR-17-92 cluster maps, associated with overexpression of its members. In this study, we address the potential role of miR-20a, from the miR-17-92 cluster, in the suppression of TGF-ß cytostatic response in CRC. Using the poorly tumorigenic and TGF-ß-sensitive FET cell line that expresses low miR-20a levels, we first confirmed that miR-20a downmodulated CDKN1A expression, both at mRNA and protein level, through direct binding to its 3'-UTR. We demonstrated that miR-20a significantly diminished cell response to TGF-ß by preventing its delay of G1/S transition and promoting progression into cell cycle. Moreover, besides modulating CDKN1A, miR-20a blocked TGF-ß-induced transactivation of its promoter without affecting the post-receptor activation of Smad3/4 effectors directly. Finally, miR-20a abrogated the TGF-ß-mediated c-Myc repression, a direct inhibitor of the CDKN1A promoter activation, most likely by reducing the expression of specific MYC-regulating genes from the Smad/E2F-based core repressor complex. Our experiments indicate that miR-20a interferes with the colonic epithelium homeostasis by disrupting the regulation of Myc/p21 by TGF-ß, which is essential for its malignant transformation.

Solitary fibrous tumors: loss of chimeric protein expression and genomic instability mark dedifferentiation.

Solitary fibrous tumors, which are characterized by their broad morphological spectrum and unpredictable behavior, are rare mesenchymal neoplasias that are currently divided into three main variants that have the NAB2-STAT6 gene fusion as their unifying molecular lesion: usual, malignant and dedifferentiated solitary fibrous tumors. The aims of this study were to validate molecular and immunohistochemical/biochemical approaches to diagnose the range of solitary fibrous tumors by focusing on the dedifferentiated variant, and to reveal the genetic events associated with dedifferentiation by integrating the findings of array comparative genomic hybridization. We studied 29 usual, malignant and dedifferentiated solitary fibrous tumors from 24 patients (including paired samples from five patients whose tumors progressed to the dedifferentiated form) by means of STAT6 immunohistochemistry and (when frozen material was available) reverse-transcriptase polymerase chain reaction and biochemistry. In addition, the array comparative genomic hybridization findings were used to profile 12 tumors from nine patients. The NAB2/STAT6 fusion was detected in all of the tumors, but immunohistochemistry and western blotting indicated that chimeric protein expression was atypical or absent in 9 out of 11 dedifferentiated tumors. The comparative genomic hybridization results revealed that the usual and malignant solitary fibrous tumors had a simple profile, whereas the genome of the dedifferentiated tumors was complex and unstable, and suggested that 13q and 17p deletions and TP53 mutations may be present in malignant lesions before the full expression of a dedifferentiated phenotype. Solitary fibrous tumor dedifferentiation is associated with the loss of chimeric oncoprotein expression, genomic instability, and cell decommitment and reprogramming. The assessment of dedifferentiated solitary fibrous tumors is based on the presence of the fusion transcripts and, in principle, negative STAT6 immunohistochemistry should not rule out a diagnosis of solitary fibrous tumor.

Gene expression profiling of circulating tumor cells in breast cancer.

BACKGROUND: Determining the transcriptional profile of circulating tumor cells (CTCs) may allow the acquisition of clinically relevant information while overcoming tumor heterogeneity-related biases associated with use of tissue samples for biomarker assessment. However, such molecular characterization is challenging because CTCs are rare and outnumbered by blood cells. METHODS: Here, we describe a technical protocol to measure the expression of >29 000 genes in CTCs captured from whole blood with magnetic beads linked with antibodies against epithelial cell adhesion molecule (EpCAM) and the carcinoma-associated mucin, MUC1, designed to be used for CTC characterization in clinical samples. Low numbers of cells (5-200) from the MCF7 and MDA-MB-468 breast cancer cell lines were spiked in healthy donor blood samples and isolated with the AdnaTest EMT-1/Stem CellSelect kit. Gene expression profiles (GEPs) were obtained with the WG-DASL HT assay and compared with GEPs obtained from RNA isolated from cultured cell lines and unspiked samples. RESULTS: GEPs from samples containing 25 or more spiked cells correlated (r = 0.95) with cognate 100-ng RNA input samples, clustered separately from blood control samples, and allowed MCF7 and MDA-MB-468 cells to be distinguished. GEPs with comparable technical quality were also obtained in a preliminary series of clinical samples. CONCLUSIONS: Our approach allows technically reliable GEPs to be obtained from isolated CTCs for the acquisition of biologically useful information. It is reproducible and suitable for application in prospective studies to assess the clinical utility of CTC GEPs, provided that >25 CTCs can be isolated.

DPD and UGT1A1 deficiency in colorectal cancer patients receiving triplet chemotherapy with fluoropyrimidines, oxaliplatin and irinotecan.

AIMS: Triplet chemotherapy with fluoropyrimidines, oxaliplatin and irinotecan is a standard therapy for metastatic colorectal cancer (CRC). Single nucleotide polymorphisms (SNPs) in DPYD and UGT1A1 influence fluoropyrimdines and irinotecan adverse events (AEs). Low frequency DPYD variants (c.1905 + 1G > A, c.1679 T > G, c.2846A > T) are validated but more frequent ones (c.496A > G, c.1129-5923C > G and c.1896 T > C) are not. rs895819 T > C polymorphism in hsa-mir-27a is associated with reduced DPD activity. In this study, we evaluated the clinical usefulness of a pharmacogenetic panel for patients receiving triplet combinations. METHODS: Germline DNA was available from 64 CRC patients enrolled between 2008 and 2013 in two phase II trials of capecitabine, oxaliplatin and irinotecan plus bevacizumab or cetuximab. SNPs were determined by Real-Time PCR. We evaluated the functional variants in DPYD (rare: c.1905 + 1G > A, c.1679 T > G, c.2846A > T; most common: c.496A > G, c.1129-5923C > G, c.1896 T > C), hsa-mir-27a (rs895819) and UGT1A1 (*28) genes to assess their association with grade 3-4 AEs. RESULTS: None of the patients carried rare DPYD variants. We found DPYD c.496A > G, c.1129-5923C > G, c.1896 T > C in heterozygosity in 19%, 5% and 8%, respectively, homozygous rs895819 in hsa-mir-27a in 9% and homozygous UGT1A1*28 in 8%. Grade 3-4 AEs were observed in 36% patients and were associated with DPYD c.496A > G (odds ratio (OR) 4.93, 95% CI 1.29, 18.87; P = 0.021) and homozygous rs895819 in hsa-mir-27a (OR 11.11, 95% CI 1.21, 102.09; P = 0.020). Carriers of DPYD c.1896 T > C and homozygous UGT1A1*28 showed an OR of 8.42 (95% CI 0.88, 80.56; P = 0.052). Multivariate analysis confirmed an independent value for DPYD c.496A > G and c.1896 T > C. CONCLUSIONS: Concomitant assessment of DPYD variants and the UGT1A1*28 allele is a promising strategy needing further validation for dose personalization.

Receptor tyrosine kinase profiles and human papillomavirus status in oropharyngeal squamous cell carcinoma.

BACKGROUND: Human papillomavirus (HPV)-positive and HPV-negative oropharyngeal squamous cell carcinomas (OSCCs) are two distinct entities. We defined the molecular profiles of druggable receptor tyrosine kinases (RTKs) in both groups. MATERIALS AND METHODS: E5 expression and RTK alterations were studied in 17 HPV-positive and 59 HPV-negative formalin-fixed OSCCs. RTK activation was explored in further 12 frozen OSCCs. RESULTS: The HPV-positive OSCCs showed E5 expression and 33.3% expressed low level of HER2. The HPV-negative OSCCs showed HER2 expression (31.2%), increased HER2 gene copy number (46.51%, P = 0.045) and HER2 activation through HER2/EGFR heterodimerisation; HER3 (51.06%, P = 0.008) and neuregulin (65.63%; P = 0.03) expression, HER3 activation and HER3/EGFR heterodimerisation; and increased IGF-1R copy number (40.50%, P = 0.021), high IGF-1R cDNA values (P = 0.002), IGF-1R activation and expression of IGF1/2 and amphiregulin. PI3KCA mutations/expression/increased gene copy number and PTEN mutations were found in both groups, whereas PTEN gene loss was only observed in the HPV-positive cases. CONCLUSION: Human papillomavirus-positive and HPV-negative OSCC showed different RTK profiles. In HPV-positive cases, it would be interesting to study the expression of E5, which may modulate EGFR turnover and activate VEGF and PDGFRß. In HPV-negative cases, HER3 may be a promising druggable biomarker that deserves further investigation. PI3KCA and PTEN alterations encourage the promising clinical evaluation of PI3K/mTOR inhibitor activity in OSCC, particularly in HPV-positive/PI3KCA-mutated OSCCs because they may be driven by PI3KCA mutation alone.

Functional characterization of NTRK1 mutations identified in melanoma.

Cutaneous melanoma is the most aggressive form of skin cancer, with a complex and heterogeneous aetiology. Deregulation of the mitogen activated protein kinase cascade is common in melanoma, due to activating mutations in the BRAF and NRAS genes. Genetic studies and high-throughput screening technologies have recently identified several somatic mutations affecting different receptor tyrosine kinase (RTK) genes. For the majority of these, however, the contribution to the complexity of melanoma biology has not been assessed. Among these, two novel missense somatic mutations (M379I and R577G) have recently been identified in the gene encoding the neurotrophic RTK NTRK1. The NTRK1 melanoma-associated point mutations were introduced in a NTRK1 expression plasmid. Functional characterization of mutants was assessed after transient and stable transfection in HeLa and NIH3T3 cells, respectively. We showed that M379I and R577G NTRK1 receptors do not display the kinase as constitutively activated and are functionally indistinguishable from the wild-type NTRK1 receptor. Our results indicate that a causative role for M379I and R577G NTRK1 mutations in melanoma development is highly unlikely. This supports the issue that, in parallel to systematic large scale cancer genome screening, functional studies are required to distinguish between mutations that play a causative role in tumor development and others that may only be passenger changes.

Axillary lymph node dissection versus no dissection in patients with T1N0 breast cancer: a randomized clinical trial (INT09/98).

BACKGROUND: Although axillary surgery is still considered to be a fundamental part of the management of early breast cancer, it may no longer be necessary either as treatment or as a guide to adjuvant treatment. The authors conducted a single-center randomized trial (INT09/98) to determine the impact of avoiding axillary surgery in patients with T1N0 breast cancer and planning chemotherapy based on biological factors of the primary tumor on long-term disease control. METHODS: From June 1998 to June 2003, 565 patients aged 30 years to 65 years with T1N0 breast cancer were randomized to either quadrantectomy with (QUAD) or without (QU) axillary lymph node dissection; a total of 517 patients finally were evaluated. All patients received radiotherapy to the residual breast only. Chemotherapy for patients in the QUAD treatment arm was determined based on lymph node status, estrogen receptor status, and tumor grade. Chemotherapy for patients in the QU treatment arm was based on estrogen receptor status, tumor grade, and human epidermal growth factor receptor 2 and laminin receptor status. Overall survival (OS) was the primary endpoint. Disease-free survival (DFS) and rate and time of axillary lymph node recurrence in the QU treatment arm were the secondary endpoints. RESULTS: After a median follow-up of >10 years, the estimated adjusted hazards ratio of the QUAD versus QU treatment arms for OS was 1.09 (95% confidence interval, 0.59-2.00; P = .783) and was 1.04 (95% confidence interval, 0.56-1.94; P = .898) for DFS. Of the 245 patients in the QU treatment arm, 22 (9.0%) experienced axillary lymph node recurrence. The median time to axillary lymph node recurrence from breast surgery was 30.0 months (interquartile range, 24.2 months-73.4 months). CONCLUSIONS: Patients with T1N0 breast cancer did not appear to benefit in terms of DFS and OS from immediate axillary lymph node dissection in the current randomized trial. The biological characteristics of the primary tumor appear adequate for guiding adjuvant treatment.

NqA: an R-based algorithm for the normalization and analysis of microRNA quantitative real-time polymerase chain reaction data.

In this note, we propose an R function named NqA (Normalization qPCR Array, where qPCR is quantitative real-time polymerase chain reaction) suitable for the identification of a set of microRNAs (miRNAs) to be used for data normalization in view of subsequent validation studies with qPCR data. NqA is available through the website of the Fondazione IRCCS Istituto Nazionale dei Tumori of Milan (http://www.istitutotumori.mi.it/modules.php?name=Content&pa=showpage&pid=812) with a dedicated user's guide. We applied our function on a qPCR dataset downloaded from the Gene Expression Omnibus (GEO) database. Results show that NqA provides a functional subset of reference miRNAs and a set of promising significantly modulated miRNAs for subsequent validation studies.

In vitro and in silico studies of MDM2/MDMX isoforms predict Nutlin-3A sensitivity in well/de-differentiated liposarcomas.

The molecular marker of well-differentiated/de-differentiated liposarcomas is MDM2 gene amplification coupled with protein overexpression and wild-type TP53. MDMX is a recently identified MDM2 homolog and its presence in this tumor is unexplored. Our aim was to investigate the role of full-length MDM2 and MDMX proteins and their isoforms in surgical specimens of well-differentiated/de-differentiated liposarcomas in view of Nutlin-3A (a MDM2 inhibitor) treatment. Frozen and matched formalin-fixed, paraffin-embedded material from surgical specimens was examined by means of: (1) fluorescence in situ hybridization to determine MDM2 and MDMX gene copy numbers; (2) RT-PCR and densitometry to analyze alternative splicing forms of mdm2 and mdmx; (3) immunoblotting and immunohistochemistry to assess the corresponding translated proteins; and (4) in vitro and in silico assays to determine their affinity for Nutlin-3A. All these cases showed MDM2 gene amplification with an MDMX disomic pattern. In all cases, the full-length mdm2 transcript was associated with the mdm2-b transcript, with ratios ranging from 0.07 to 5.6, and both were translated into protein; mdmx and mdmx-s were co-transcripted, with ratios ranging from 0.1 to 5.6. MDMX-S was frequently more upregulated than MDMX at both transcriptional and protein level. Each case showed different amounts of mdm2, mdm2-b, mdmx, and mdmx-s transcripts and the corresponding proteins. In vitro assays showed that Nutlin-3A was ineffective against MDM2-B and was unable to disrupt the MDMX/TP53 and MSMX-S/TP53 complexes. Molecular simulations confirmed these in vitro findings by showing that MDM2 has high Nutlin-3A affinity, followed by MDMX-S, MDMX, and MDM2-B. Nutlin-3A is predicted to be a good therapeutic option for well-differentiated/de-differentiated liposarcomas. However, our findings predict heterogeneous responses depending on the relative expression of mdm2, mdm2-b, mdmx, and mdmx-s transcripts and proteins.

Receptor tyrosine kinase pathway analysis sheds light on similarities between clear-cell sarcoma and metastatic melanoma.

To highlight possible similarities and differences in receptor tyrosine kinase (RTK) and downstream signalling activation profiles between clear-cell sarcomas (CCS) and metastatic melanomas (MM), frozen, and paired-matched fixed samples of six CCS with EWSR1 rearrangement (EWSR1+), five CCS without EWSR1 rearrangement (EWSR1-), and seven MM were investigated by means of biochemical, immunohistochemical, FISH, molecular analyses, and immunofluorescence confocal microscopy. Fixed samples of a further 10 CCS and 14 MM were investigated by means of sequencing for BRAF, NRAS, and KRAS mutations and FISH analyses for the gain of chromosomes 22 and 8. RTK analysis of all CCS/MM samples showed activation of short-form (sf) recepteur d'origine nantais (RON) RTK and of PDGFRB, MET, and HER3. Analysis of downstream signaling revealed consistent phosphorylation patterns of PI3K/AKT, RSK, and the mTOR targets S6 and 4EBP1. Analysis of frozen and fixed material from 21 CCS and 21 MM showed the presence of the V600E BRAF mutation in 2/12 EWSR1+ and 3/9 EWSR1- CCS and 9/21 MM and demonstrated a significant (P < 0.001) correlation between the gain of chromosomes 22 and 8 and EWSR1- CCS. Our results show that BRAF mutation can also be present in CCS and support the proposed aberration of chromosomes 22 and 8 as a possibly useful nonrandom hallmark of EWSR1- CCS. Besides, they broaden the spectrum of the similarities of RTK pathway activation between CCS and MM, thus suggesting that new drugs found to be active in melanoma and RON inhibitors could have a role in CCS treatment. © 2011 Wiley Periodicals, Inc.

NF1 Gene Inactivation Acts as Tumor Driver in RET/RAS Negative Medullary Thyroid Carcinomas.

OBJECTIVE: 20% of sporadic MTC has no RET/RAS somatic alterations or other known gene alterations. Aim of this study was to investigate RET/RAS negative MTC for the presence of NF1 alterations. METHODS: we studied 18 sporadic RET/RAS negative MTC cases: Next generation sequencing of tumoral and blood DNA was performed using a custom panel including the entire coding region of the NF1 gene. The effect of NF1 alterations on the transcripts were characterized by RT-PCR and the loss of heterozygosity of the other NF1 allele was investigated with Multiplex Ligation-dependent Probe Amplification. RESULTS: Two cases showed bi-allelic inactivation of NF1 with a prevalence of about 11% of RET/RAS negative cases. In a patient affected by neurofibromatosis there was a somatic intronic point mutation determining the transcript alteration in one allele and a germline loss of heterozygosity (LOH) in the other. In the other case described both the point mutation and the LOH were somatic events; this latter finding shows, for the first time, a driver role of NF1 inactivation in MTC independent of RET/RAS alterations and the presence of neurofibromatosis. CONCLUSIONS: About 11% of our series of sporadic RET/RAS negative MTC harbor biallelic inactivation of NF1 suppressor gene also regardless neurofibromatosis status. According to our results, NF1 alterations should be searched in all RET/RAS negative MTC as possible driver. Moreover, this finding reduces the number of negative sporadic MTCs and may have important clinical implications in the management of these tumors.

Decellularized extracellular matrix as scaffold for cancer organoid cultures of colorectal peritoneal metastases.

Peritoneal metastases (PM) from colorectal cancer (CRC) are associated with poor survival. The extracellular matrix (ECM) plays a fundamental role in modulating the homing of CRC metastases to the peritoneum. The mechanisms underlying the interactions between metastatic cells and the ECM, however, remain poorly understood, and the number of in vitro models available for the study of the peritoneal metastatic process is limited. Here, we show that decellularized ECM of the peritoneal cavity allows the growth of organoids obtained from PM, favoring the development of three-dimensional (3D) nodules that maintain the characteristics of in vivo PM. Organoids preferentially grow on scaffolds obtained from neoplastic peritoneum, which are characterized by greater stiffness than normal scaffolds. A gene expression analysis of organoids grown on different substrates reflected faithfully the clinical and biological characteristics of the organoids. An impact of the ECM on the response to standard chemotherapy treatment for PM was also observed. The ex vivo 3D model, obtained by combining patient-derived decellularized ECM with organoids to mimic the metastatic niche, could be an innovative tool to develop new therapeutic strategies in a biologically relevant context to personalize treatments.

OncoPan®: An NGS-Based Screening Methodology to Identify Molecular Markers for Therapy and Risk Assessment in Pancreatic Ductal Adenocarcinoma.

Pancreatic cancer has a high morbidity and mortality with the majority being PC ductal adenocarcinomas (PDAC). Whole genome sequencing provides a wide description of genomic events involved in pancreatic carcinogenesis and identifies putative biomarkers for new therapeutic approaches. However, currently, there are no approved treatments targeting driver mutations in PDAC that could produce clinical benefit for PDAC patients. A proportion of 5-10% of PDAC have a hereditary origin involving germline variants of homologous recombination genes, such as Mismatch Repair (MMR), STK11 and CDKN2A genes. Very recently, BRCA genes have been demonstrated as a useful biomarker for PARP-inhibitor (PARPi) treatments. In this study, a series of 21 FFPE PDACs were analyzed using OncoPan®, a strategic next-generation sequencing (NGS) panel of 37 genes, useful for identification of therapeutic targets and inherited cancer syndromes. Interestingly, this approach, successful also on minute pancreatic specimens, identified biomarkers for personalized therapy in five PDAC patients, including two cases with HER2 amplification and three cases with mutations in HR genes (BRCA1, BRCA2 and FANCM) and potentially eligible to PARPi therapy. Molecular analysis on normal tissue identified one PDAC patient as a carrier of a germline BRCA1 pathogenetic variant and, noteworthy, this patient was a member of a family affected by inherited breast and ovarian cancer conditions. This study demonstrates that the OncoPan® NGS-based panel constitutes an efficient methodology for the molecular profiling of PDAC, suitable for identifying molecular markers both for therapy and risk assessment. Our data demonstrate the feasibility and utility of these NGS analysis in the routine setting of PDAC molecular characterization.

Exploring the association with disease recurrence of miRNAs predictive of colorectal cancer.

INTRODUCTION: Disease recurrence after surgery is a crucial predictor of poor prognosis in colorectal cancer, where disseminated disease at the time of intervention can also be observed in localized early-stage cases. We evaluated the ability to predict disease recurrence of miRNAs from two signatures that we have found linked to the presence of colorectal cancer (CL signature) or adenoma (HgA signature) in higher-risk subjects. METHODS: miRNAs from the signatures were studied longitudinally by quantitative real-time polymerase chain reaction in plasma from 24 patients with resectable colorectal cancer collected at the time of surgery and during scheduled follow-up across 36 months. Patients either showed relapse within 36 months (alive with disease (AWD)), or remained disease-free (no evidence of disease (NED)) for the same period. RESULTS: Although the signatures did not predict recurrence, expression of the miRNAs from the CL signature decreased 1 year after surgery, and one miRNA of the signature, miR-378a-3p, almost reached significance in the NED subgroup (Wilcoxon signed-rank test: p-value = 0.078). Also, miR-335-5p from the HgA signature was higher in AWD patients before surgery (Kruskal-Wallis test: p-value = 0.019). CONCLUSIONS: These data, although from a small cohort of patients, support the possible use of miRNAs as non-invasive biomarkers in liquid biopsy-based tests to identify patients at risk of relapse and to monitor them during follow-up.

Transcriptional characteristics of familial non-BRCA1/BRCA2 breast tumors.

To better understand the alterations present in the group of the so-called BRCAX tumors, we have used a cDNA microarray containing genes related to tumorigenesis and analyzed a series of 49 tumors consisting of 13 BRCA1, 14 BRCAX and 22 sporadic. We have confirmed that the BRCAX tumors are heterogeneous and can be divided in at least two main subgroups, so-called A and B, transcriptionally distinguishable and with different altered pathways within each of the groups. We have found that BRCAX-A and B subgroups, can be classified as Luminal A and Luminal B, respectively, taking into account the intrinsic phenotypes defined for the sporadic breast tumors. We have found that, at the somatic level, the BRCAX-B tumors are identical to their sporadic Luminal B counterparts, whereas BRCAX-A, despite having a Luminal A phenotype, shows additional genomic alterations. We have found 21 deregulated genes in the BRCAX-A group that we have called "the BRCAX susceptibility pathway" and suggested it as a candidate to search for new genes involved in the inherited susceptibility underlying the disease in this group.

Gene expression analysis reveals a different transcriptomic landscape in female and male breast cancer.

Male breast cancer (MBC) is a poorly characterized disease because of its rarity. Clinical management is based on results obtained from randomized trials conducted in women notwithstanding data in the literature suggesting relevant gender-associated differences in terms of biological and clinical behavior. However, a genome-wide characterization of MBC on a transcriptional level is lacking. In this study, gene expression profiles of 37 estrogen receptor positive (ER+) MBC specimens were compared to that of 53 ER+ Female Breast Cancer (FBC) samples similar for clinical and patho-biological features. Almost 1000 genes were found differentially expressed (FDR < 1%) between female and male patients and biological interpretation highlighted a gender-associated modulation of key biological processes ranging from energy metabolism to regulation of translation and matrix remodeling as well as immune system recruitment. Moreover, an analysis of genes correlated to steroid receptors and ERBB2 suggested a prominent role for the androgen receptor in MBC with a minor relevance for progesterone receptor and ERBB2, although, similarly to FBC, a genomic amplification could be observed. Our findings support the idea that breast cancer is a quite different disease in male and female patients and the underlying gender-related biological differences are likely to have clinical implications connected with different susceptibility to treatment.

Proteomic detection of a large amount of SCGFα in the stroma of GISTs after imatinib therapy.

BACKGROUND: Gastrointestinal stromal tumors (GISTs) are the most frequent mesenchymal tumors to develop in the digestive tract. These tumors are highly resistant to conventional chemotherapy and only the introduction of imatinib mesylate has improved the prognosis of patients. However, Response Evaluation Criteria in Solid Tumors are inappropriate for assessing tumor response, and the histological/pathological response to imatinib is variable, heterogeneous, and does not associate with clinical response. The effects of imatinib on responding GISTs are still being explored, and few studies correlate the clinical response with the histological response after pharmacological treatment. Recently, apoptosis and autophagy were suggested as possible alternative mechanisms of pharmacological response. METHODS: Here, we used a proteomic approach, combined with other analyses, to identify some molecular stromal components related to the response/behavior of resected, high-risk GISTs after neoadiuvant imatinib therapy. RESULTS: Our proteomic results indicate an elevated concentration of Stem Cell Growth Factor (SCGF), a hematopoietic growth factor having a role in the development of erythroid and myeloid progenitors, in imatinib-responsive tumor areas. SCGFα expression was detected by mass spectrometry, immunohistochemistry and/or western blot and attributed to acellular matrix of areas scored negative for KIT (CD117). RT-PCR results indicated that GIST samples did not express SCGF transcripts. The recently reported demonstration by Gundacker et al. 1 of the secretion of SCGF in mature pro-inflammatory dendritic cells would indicate a potential importance of SCGF in tissue inflammatory response. Accordingly, inflammatory infiltrates were detected in imatinib-affected areas and the CD68-positivity of the SCGF-positive and KIT-negative areas suggested previous infiltration of monocytes/macrophages into these regions. Thus, chronic inflammation subsequent to imatinib treatment may determine monocyte/macrophage recruitment in imatinib-damaged areas; these areas also feature prominent tumor-cell loss that is replaced by dense hyalinization and fibrosis. CONCLUSIONS: Our studies highlight a possible role of SCGFα in imatinib-induced changes of GIST structure, consistent with a therapeutic response.

CIGNOweb.it.

We introduce CIGNOweb.it, a database of oncology resources for patients, the general public and healthcare professionals. It builds on the previous Italian cancer resource Azaleaweb and offers quality-evaluated content. It meets international bibliographic and technical standards such as the Open Archives Initiative (OAI) for web content interoperability and the Functional Requirements for Bibliographic Records (FRBR) for bibliographic description with respect to the different media, applications, and user needs. Database content is supplied in collaboration with non-profit associations, libraries and the network of Cancer Information Points that is currently being established all over Italy. Expert and customer evaluation and feedback are provided for in the system. The graphic layout has been painstakingly designed to be user-friendly for a non-expert public. CIGNOweb.it is multicentric and will in time offer health information outside the field of oncology. It is designed to become a multilingual tool to organize, optimize and access patient information produced in the languages of the "newer" European countries. It is hoped that CIGNOweb.it will support other European nations in enhancing the structure and organization of their own-language patient health information and will contribute towards making a common health information portal of the European Union a reality.

Chromosome band 17q21 in breast cancer: significant association between beclin 1 loss and HER2/NEU amplification.

Treatment success of breast cancer patients with trastuzumab alone or in combination depends not only on HER2/NEU amplification but also on PTEN and PI3K status and efficient cell death programs. In this pilot study, we found a significant association between loss of beclin 1 and HER2/NEU amplification (both on 17q21) in breast cancers. This finding was confirmed in two public copy number microarray datasets. Furthermore, there is a trend associating beclin 1 loss with TP53 mutations, PI3KCA gene gain, and PTEN mutations. Finally, the observation that beclin 1 gene loss predicted a response to trastuzumab alone or in combination with other drugs is worthy of further confirmation in larger cohorts. Our results suggest that, beclin 1 loss may contribute to genome instability and to a defective autophagy that may lead to tumoral cell death in presence of competent apoptosis or senescence pathways.