SPIDIA-RNA: First external quality assessment for the pre-analytical phase of blood samples used for RNA based analyses

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A three-gene signature marks the time to locoregional recurrence in luminal-like breast cancer

Background: Gene expression profiling (GEP)-based prognostic signatures are being rapidly integrated into clinical decision making for systemic management of breast cancer patients. However, GEP remains relatively underdeveloped for locoregional risk assessment. Yet, locoregional recurrence (LRR), especially early after surgery, is associated with poor survival. Patients and methods: GEP was carried out on two independent luminal-like breast cancer cohorts of patients developing early (≤5 years after surgery) or late (>5 years) LRR and used, by a training and testing approach, to build a gene signature able to intercept women at risk of developing early LRR. The GEP data of two in silico datasets and of a third independent cohort were used to explore its prognostic value. Results: Analysis of the first two cohorts led to the identification of three genes, CSTB, CCDC91 and ITGB1, whose expression, derived by principal component analysis, generated a three-gene signature significantly associated with early LRR in both cohorts (P value <0.001 and 0.005, respectively), overcoming the discriminatory capability of age, hormone receptor status and therapy. Remarkably, the integration of the signature with these clinical variables led to an area under the curve of 0.878 [95% confidence interval (CI) 0.810-0.945]. In in silico datasets we found that the three-gene signature retained its association, showing higher values in the early relapsed patients. Moreover, in the third additional cohort, the signature significantly associated with relapse-free survival (hazard ratio 1.56, 95% CI 1.04-2.35). Conclusions: Our three-gene signature represents a new exploitable tool to aid treatment choice in patients with luminal-like breast cancer at risk of developing early recurrence

Identification of microRNAs for the early diagnosis of colorectal cancer (CRC)

Introduction: microRNAs (miRNAs) are small non-coding RNAs involved in the development of various cancers. Quantitative real-time PCR (qPCR) is the assay commonly used to investigate miRNA expression and qPCR-low-density arrays are the most used technique for both identification and validation of modulated miRNAs. One crucial pre-processing step for miRNA analysis is data normalization, aimed at reducing nonbiological sources of variation [1]. This process would allow to identify a small set of miRNAs to be used for data normalization in subsequent validation studies [2]. Materials and methods: we analyzed the expression levels of 381 human miRNAs on TaqMan Array MicroRNA Card A v.2 (Applied Biosystems), on a cohort of 60 plasma samples (38 precancerous lesions/cancer and 22 without lesion) from individuals enrolled in the CRC screening program of the Milan Local Health Authority that underwent colonoscopy at our Institute (INT) after a positive fecal occult blood test (FIT+). Starting from these data, we developed and applied a data-driven normalization method able to identify a small set of reference miRNAs to use for data normalization in the subsequent validation studies [2,3]. Briefly, by considering the miRNAs expressed in all the samples, the relative expression of each miRNA was first computed according to their mean expression value [4] and the best subset of miRNAs that resemble this value was selected [2,3]. Results and discussion: we identified 4 housekeeping miRNAs suitable for data normalization and 7 miRNAs significantly different in subjects with precancerous/cancerous lesions versus subjects without lesions. We also identified 4 miRNAs related to presence of initial adenoma, one linked to advanced adenoma and 8 to presence of cancerous lesion. We are now constructing of a Custom TaqMan Array Cards including the identified miRNAs and some other miRNAs of interest, i.e those haemolysis-related and miR-378 [5], with the aim of validating these biomarkers on a prospective cohort of 120 FIT+ subjects that underwent colonoscopy at INT. Conclusion: the adopted strategy allowed the identification of reference miRNAs to be used for data normalization and the identification of modulated miRNAs that will be validated in larger prospective series. Acknowledgements: This work was supported by grants from Associazione Italiana per la Ricerca sul Cancro (AIRC) (Grants No.10529 and No. 12162 to MA Pierotti)

A Comparison Between Conventional Formalin and Novel PAXgene Tissue Fixation: the European Spidia Morphology Ring Trials Experience

Introduction and Aim: Molecular pathology is an emerging discipline requiring high quality tissue samples which allow simultaneous molecular and histopathological analysis. Within the European FP7 project (grant agreement no. 222916) \u201cStandardisation and improvement of generic pre-analytical tools and procedures for in-vitro diagnostics\u201d (SPIDIA) different morphology ring trials were implemented. The aim of these ring trials was to evaluate the quality of histomorphology and suitability of PAXgene-fixed and paraffin-embedded (PFPE) samples for routine diagnostics, with special emphasis on the tumor grading, in comparison to the current state-of-the-art technique for routine morphological diagnostics formaldehyde-fixed and paraffin-embedded (FFPE) samples in breast and colon cancer tissues. Methods: Each cancer tissue sample was divided into two mirrored samples for PFPE and FFPE. Haematoxylin and eosin and Periodic-Acid-Schiff stained sections were scanned and evaluated in a blinded, randomized ring trial by pathologists from Europe and the US using virtual microscopy. Each participant evaluated only one of the two virtual slides obtained from each case according to a randomized scheme implying the subdivision of both, cases and participants, in two groups. Links to the randomized virtual slides were distributed by email to the participants together with the study protocol, the electronic evaluation form and all instructions and timeline for the ring trial implementation. The reproducibility between fixation methods (PFPE vs. FFPE) was assessed by computing the modal category within each method for each case (Modal Scenario, MS). By starting from these values, the jackknifed estimate of the weighted kappa statistic (Kwj)1 was computed together with the relative 95% Confidence Interval (CI)2. In addition the inter-observer reproducibility was assessed by computing the Kwj and the kappa category-specific statistics (Kcs) and their weighted average (Cohen\u2019s kappa statistic, Kc) were estimated by jointly considering all participant\u2019s data1,3. Results: Tissue samples of 18 breast and 15 colon cancer cases were evaluated by 23 and 20 pathologists, respectively. By using the MS, a high and quite satisfactory level of reproducibility between PFPE and FFPE samples in evaluating the histological grading score was observed in breast (kwj=0.92, 95% CI: 0.76-1.00) and colon (kwj=0.73, 95% CI: 0.41-0.94) cancer, respectively. Moreover the inter-observer reproducibility for grading was not completely satisfactory in both ring trials, with G2 as the most critical category, but showed a similar performance level in each of the two considered groups of pathologists. Conclusions: Our findings suggest that the methodological approach we developed is suitable for evaluating the reproducibility between alternative fixation methods in the ring trial setting implemented here and that histomorphology is excellently preserved in PFPE tissues. References: 1. Fleiss JL. 2nd Ed. New York: Wiley and Sons 1981. 2. Corletto V, Verderio P, Giardini R et al. Anal Cell Pathol. 1998;16:83-93. 3. Holman CD. Am J Epidemiol. 1984;120:154-60. 4. Landis JR, Koch GG. Biometrics. 1977;33:159-74

PI3KCA mutation status is of limited prognostic relevance in ER-positive breast cancer patients treated with hormone therapy

PI3K/AKT/mTOR pathway alterations are frequent in patients with infiltrating breast cancer (IBC). Their clinical and pathological relevance has been insufficiently documented. We evaluated PI3KCA for mutations and the expression of PTEN, AKT, mTOR and p70S6K by immunohistochemistry in 246 IBC patients treated with hormone therapy (median follow-up, 97 months). A PI3KCA mutation was observed in 50 out of 229 informative cases (21.8 %), PTEN loss in 107 out of 210 (51 %), moderate/high level of expression of AKT in 133 out of 188 (71 %), moderate/high level of expression of mTOR in 173 out of 218 (79 %) and moderate/high level of expression of p70S6K in 111 out of 192 cases (58 %). PI3KCA mutation was associated with the absence of Her2/ neu amplification/overexpression and a low level of MIB1/Ki- 67 labelling. The expression of p70S6K was associated with a high level of mTOR immunoreactivity, and high PTEN expression was associated with high AKT expression level. Univariate analysis showed that PI3KCA mutation status was not associated with clinical outcome in the series as a whole or in the node-negative subgroup. However, in the node-positive subgroup, exon 9 PI3KCA mutation was associated with unfavourable overall survival (OS), although its impact on the final model in multivariate analysis seemed to be limited. Of the other markers, only high p70S6K expression was associated with a significantly prolonged OS. PI3KCA mutation status is of limited prognostic relevance in oestrogen receptor-positive breast cancer patients treated with hormone therapy