Sara Pizzamiglio

Evaluation of SNPs in miR-146a, miR196a2 and miR-499 as low-penetrance alleles in German and Italian familial breast cancer cases

Recently, the SNPs rs11614913 in hsa‐mir‐196a2 and rs3746444 in hsa‐mir‐499 were reported to be associated with increased breast cancer risk, and the SNP rs2910164 in hsa‐mir‐146a was shown to have an effect on age of breast cancer diagnosis. In order to further investigate the effect of these SNPs, we genotyped a total of 1894 breast cancer cases negative for disease‐causing mutations or unclassified variants in BRCA1 and BRCA2, and 2760 controls from Germany and Italy. We compared the genotype and allele frequencies of rs2910164, rs11614913 and rs3746444 in cases versus controls of the German and Italian series, and of the two series combined; we also investigated the effect of the three SNPs on age at breast cancer diagnosis. None of the performed analyses showed statistically significant results. In conclusion, our data suggested lack of association between SNPs rs2910164, rs11614913 and rs3746444 and breast cancer risk, or age at breast cancer onset.

miRNA Profiling in Colorectal Cancer Highlights miR-1 Involvement in MET-Dependent Proliferation

Altered expression of miRNAs is associated with development and progression of various human cancers by regulating the translation of oncogenes and tumor suppressor genes. In colorectal cancer, these regulators complement the Vogelstein multistep model of pathogenesis and have the potential of becoming a novel class of tumor biomarkers and therapeutic targets. Using quantitative real-time PCR, we measured the expression of 621 mature miRNAs in 40 colorectal cancers and their paired normal tissues and identified 23 significantly deregulated miRNAs. We subsequently evaluated their association with clinical characteristics of the samples and presence of alterations in the molecular markers of colorectal cancer progression. Expression levels of miR-31 were correlated with CA19-9 and miR-18a, miR-21, and miR-31 were associated with mutations in APC gene. To investigate the downstream regulation of the differentially expressed miRNAs identified, we integrated putative mRNA target predictions with the results of a meta-analysis of seven public gene expression datasets of normal and tumor samples of colorectal cancer patients. Many of the colorectal cancer deregulated miRNAs computationally mapped to targets involved in pathways related to progression. Here one promising candidate pair (miR-1 and MET) was studied and functionally validated. We show that miR-1 can have a tumor suppressor function in colorectal cancer by directly downregulating MET oncogene both at RNA and protein level and that reexpression of miR-1 leads to MET-driven reduction of cell proliferation and motility, identifying the miR-1 downmodulation as one of the events that could enhance colorectal cancer progression. Mol Cancer Res; 10(4); 504–15. ©2012 AACR.

Circulating miR-378 in plasma: a reliable, haemolysis-independent biomarker for colorectal cancer

Background:Plasma circulating tumour-specific microRNAs (miRNAs) are promising biomarkers of tumour presence and recurrence, especially for diseases whose best chance of successful treatment requires early diagnosis and timely surgery of an already malignant but not yet invasive tumour, such as colorectal cancer (CRC).Methods:Expression levels of miRNAs previously found to be differently expressed in tumour vs normal colon tissues were investigated by quantitative real-time PCR in plasma from CRC patients and from healthy donors and confirmed in independent case control series. The validated miRNAs were also measured after surgery. Analyses were repeated on the subsets of haemolysis-free samples.Results:We identified four miRNAs differently expressed between the compared groups, two (miR-21 and miR-378) of which were validated. miR-378 expression decreased in non-relapsed patients 4–6 months after surgery and miR-378 ability to discriminate CRC patients from healthy individuals was not influenced by haemolysis levels of plasma samples.Conclusion:The miRNA analysis on plasma samples represents a useful non-invasive tool to assess CRC presence as well as tumour-free status at follow-up. Plasma levels of miR-378 could be used to discriminate CRC patients from healthy individuals, irrespective of the level of haemoglobin of plasma samples.

Tumor response assessment by modified Choi criteria in localized high-risk soft tissue sarcoma treated with chemotherapy.

The objective of this study was to compare the prognostic relevance of Response Evaluation Criteria in Solid Tumors (RECIST) versus Choi criteria for the assessment of response in patients with high‐risk soft tissue sarcoma of the extremities or trunk wall who received preoperative chemotherapy with or without radiotherapy in a phase 3 trial.

Quality of surgery and neoadjuvant combined therapy in the ISG-GEIS trial on soft tissue sarcomas of limbs and trunk wall

BACKGROUND To explore correlation between the quality of surgery and outcome in high-risk soft tissue sarcoma (STS) patients treated within a phase III randomized trial. PATIENTS AND METHODS In the trial, all patients received three cycles of preoperative chemotherapy (CT) with epirubicin 120 mg/m(2) and ifosfamide 9 g/m(2) and were randomly assigned to receive two further postoperative cycles. Radiotherapy (RT) could be delivered in the preoperative or postoperative setting. The association between surgical margins and overall survival (OS) was studied in a univariate and multivariate fashion. RESULTS Two hundred and fifty-two patients completed the whole treatment and were operated conservatively. At a median follow-up of 60 months (IQR, 45-74 months), the 5-year OS was 0.73, even in patients with positive and negative margins. The 5-year cumulative incidence (CI) of local recurrence (LR) in patients with positive and negative microscopic margins was 0.17 (standard error, SE, 0.08) and 0.03 (SE, 0.01), respectively. In the subgroup of patients receiving combined preoperative CT-RT and with positive surgical margins, the CI of LR was 0. CONCLUSIONS In this setting of high-risk STS treated by preoperative CT or CT-RT, the negative impact of positive margins on the outcome was limited. When close margins can be anticipated preoperative CT-RT may be a reasonable option to maximize the chance of cure.

SPIDIA-RNA: Second External Quality Assessment for the Pre-Analytical Phase of Blood Samples Used for RNA Based Analyses

One purpose of the EC funded project, SPIDIA, is to develop evidence-based quality guidelines for the pre-analytical handling of blood samples for RNA molecular testing. To this end, two pan-European External Quality Assessments (EQAs) were implemented. Here we report the results of the second SPIDIA-RNA EQA. This second study included modifications in the protocol related to the blood collection process, the shipping conditions and pre-analytical specimen handling for participants. Participating laboratories received two identical proficiency blood specimens collected in tubes with or without an RNA stabilizer. For pre-defined specimen storage times and temperatures, laboratories were asked to perform RNA extraction from whole blood according to their usual procedure and to return extracted RNA to the SPIDIA facility for further analysis. These RNA samples were evaluated for purity, yield, integrity, stability, presence of interfering substances, and gene expression levels for the validated markers of RNA stability: FOS, IL1B, IL8, GAPDH, FOSB and TNFRSF10c. Analysis of the gene expression results of FOS, IL8, FOSB, and TNFRSF10c, however, indicated that the levels of these transcripts were significantly affected by blood collection tube type and storage temperature. These results demonstrated that only blood collection tubes containing a cellular RNA stabilizer allowed reliable gene expression analysis within 48 h from blood collection for all the genes investigated. The results of these two EQAs have been proposed for use in the development of a Technical Specification by the European Committee for Standardization.

FANCM c.5791C>T nonsense mutation (rs144567652) induces exon skipping, affects DNA repair activity and is a familial breast cancer risk factor

Numerous genetic factors that influence breast cancer risk are known. However, approximately two-thirds of the overall familial risk remain unexplained. To determine whether some of the missing heritability is due to rare variants conferring high to moderate risk, we tested for an association between the c.5791C>T nonsense mutation (p.Arg1931*; rs144567652) in exon 22 of FANCM gene and breast cancer. An analysis of genotyping data from 8635 familial breast cancer cases and 6625 controls from different countries yielded an association between the c.5791C>T mutation and breast cancer risk [odds ratio (OR) = 3.93 (95% confidence interval (CI) = 1.28-12.11; P = 0.017)]. Moreover, we performed two meta-analyses of studies from countries with carriers in both cases and controls and of all available data. These analyses showed breast cancer associations with OR = 3.67 (95% CI = 1.04-12.87; P = 0.043) and OR = 3.33 (95% CI = 1.09-13.62; P = 0.032), respectively. Based on information theory-based prediction, we established that the mutation caused an out-of-frame deletion of exon 22, due to the creation of a binding site for the pre-mRNA processing protein hnRNP A1. Furthermore, genetic complementation analyses showed that the mutation influenced the DNA repair activity of the FANCM protein. In summary, we provide evidence for the first time showing that the common p.Arg1931* loss-of-function variant in FANCM is a risk factor for familial breast cancer.

Identification of fifteen novel germline variants in the BRCA1 3′UTR reveals a variant in a breast cancer case that introduces a functional miR-103 target site†

Mutations in the BRCA1 gene confer a substantial increase in breast cancer risk, yet routine clinical genetic screening is limited to the coding regions and intron–exon boundaries, precluding the identification of mutations in noncoding and untranslated regions (UTR). As 3′UTR mutations can influence cancer susceptibility by altering protein and microRNA (miRNA) binding regions, we screened the BRCA1 3′UTR for mutations in a large series of BRCA‐mutation negative, population and clinic‐based breast cancer cases, and controls. Fifteen novel BRCA1 3′UTR variants were identified, the majority of which were unique to either cases or controls. Using luciferase reporter assays, three variants found in cases, c.*528G>C, c.*718A>G, and c.*1271T>C and four found in controls, c.*309T>C, c.*379G>A, c.*823C>T, and c.*264C>T, reduced 3′UTR activity (P < 0.02), whereas two variants found in cases, c.*291C>T and c.*1139G>T, increased 3′UTR activity (P < 0.01). Three case variants, c.*718A>G, c.*800T>C, and c.*1340_1342delTGT, were predicted to create new miRNA binding sites and c.*1340_1342delTGT caused a reduction (25%, P = 0.0007) in 3′UTR reporter activity when coexpressed with the predicted targeting miRNA, miR‐103. This is the most comprehensive identification and analysis of BRCA1 3′UTR variants published to date. Hum Mutat 33:1665–1675, 2012.

Influence of storage conditions and extraction methods on the quantity and quality of circulating cell-free DNA (ccfDNA): the SPIDIA-DNAplas External Quality Assessment experience

Abstract Background: Circulating cell-free DNA (ccfDNA) has been confirmed as a useful biomarker in cancer and pre-natal clinical practice. One of the main critical points in using ccfDNA is a lack of standardisation for sample processing methods, storage conditions, procedures for extraction, and quantification that can affect ccfDNA quality and quantity. We report the results obtained from the SPIDIA-DNAplas, one of the EU SPIDIA (Standardisation and improvement of generic pre-analytical tools and procedures for in vitro diagnostics) subprojects based on the implementation of an External Quality Assessment scheme for the evaluation of the influence of the pre-analytical phase on ccfDNA. This is the first reported quality control scheme targeting ccfDNA for pre-analytical phase studies. Methods: Fifty-six laboratories throughout Europe were recruited. The participating laboratories received the same plasma sample and extracted ccfDNA by using their own procedures, at defined plasma storage conditions, and sent the isolated ccfDNA to the SPIDIA facility for analyses. Laboratory performance was evaluated by using specific quality parameters such as ccfDNA integrity (by multiplex PCR) and yield (by qPCR). Results: The analysis of the ccfDNA extracted by the laboratories showed that most of them (53 of 56) were able to recover ccfDNA but only 12.5% recovered non-fragmented ccfDNA. Extraction methods specifically designed for ccfDNA preserved the integrity profile. Conclusions: The evidence-based results of the SPIDIA-DNAplas EQA have been proposed as a basis for the development of a Technical Specification by the European Committee for standardisation (CEN).

The SNP rs895819 in miR-27a is not associated with familial breast cancer risk in Italians

MicroRNAs (miRNAs) are a class of small non-coding RNA implicated in gene expression; deregulation of miRNAs, accounted by somatic variations, may lead to initiation and progression of several types of cancers [1]. It has been postulated that also common germline variants located within miRNA genes may be associated with cancer risk. In particular, the single nucleotide polymorphisms (SNPs) rs2910164, rs11614913, rs3746444, and rs6505162 located within miR-146a, miR-196a2, miR-499, and miR423, respectively, resulted all associated with breast cancer risk [2, 3]. However, a subsequent large study on SNPs in miR-146a, miR-192a2, and miR-499 did not replicate these findings [4]. Two following meta-analyses confirmed the association for the SNP in miR-196a2 and the lack of association for the SNP in miR-146a [5, 6]. The rare allele of the rs12975333 in miRNA-125a was found strongly associated with breast cancer risk in Antwerp, Belgium [7], but a multicenter study reported no allele carriers failing to confirm this association [8]. Recently, the SNP rs895819 located in the miRNA-27a gene was investigated in a series of 1,217 German familial breast cancer cases and 1,422 unrelated German controls and the rare [G] allele was shown to have a protective effect with odds ratio (OR) = 0.88 [95 % confidence interval (CI) 0.78–0.99, P = 0.0287]. Additional analyses indicated that the protective effect was limited to cases with age at diagnosis \50 years (OR = 0.83, 95 % CI 0.70–0.98, P = 0.0314), whereas a stronger effect was detected in bilateral breast cancer cases (OR = 0.70, 95 % CI 0.52–0.95, P = 0.0238) [9]. Aimed at re-testing these findings, we investigated the effect of rs895819 on breast cancer risk by genotyping a