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Background As neoadjuvant chemotherapy (NAC) is increasingly used in triple-negative breast cancer (TNBC), we investigated the value of circulating tumor DNA (ctDNA) for patient monitoring prior, during, and after NAC, and circulating tumor cells (CTCs) for disease characterization at clinical progression. Materials and methods Forty-two TNBC patients undergoing NAC were prospectively enrolled. Primary tumor mutations identified by targeted-gene sequencing were validated and tracked in 168 plasma samples longitudinally collected at multiple time-points by droplet digital polymerase chain reaction. At progression, plasma DNA underwent direct targeted-gene assay, and CTCs were collected and analyzed for copy number alterations (CNAs) by low-pass whole genome sequencing. Results ctDNA detection after NAC was associated with increased risk of relapse, with 2-year event-free survival estimates being 44.4% [95% confidence interval (CI) 21.4%-92.3%] versus 77.4% (95% CI 57.8%-100%). ctDNA prognostic value remained worthy even after adjusting for age, residual disease, systemic inflammatory indices, and Ki-67 [hazard ratio (HR) 1.91; 95% CI 0.51-7.08]. During follow-up, ctDNA was undetectable in non-recurrent cases with the unique exception of one showing a temporary peak over eight samples. Conversely, ctDNA was detected in 8/11 recurrent cases, and predated the clinical diagnosis up to 13 months. Notably, recurrent cases without ctDNA developed locoregional, contralateral, and bone-only disease. At clinical progression, CTCs presented chromosome 10 and 21q CNAs whose network analysis showed connected modules including HER/PI3K/Ras/JAK signaling and immune response. Conclusion ctDNA is not only associated with but is also predictive of prognosis in TNBC patients receiving NAC, and represents an exploitable tool, either alone or with CTCs, for personalized TNBC management., Highlights • ctDNA was detected in 77% of early-stage TNBC patients undergoing neoadjuvant chemotherapy. • Patients with still detectable ctDNA after NAC were more than twice as likely to relapse as those with undetectable levels. • Detection of ctDNA during follow-up antedated clinical overt metastases up to 13 months. • ctDNA was undetectable in all but one non-recurrent patient with a temporary peak in only 1 of 8 samples tested. • CTCs of progressing cases lacked epithelial surface markers and showed therapeutically exploitable molecular features.

BACKGROUND: As a neoadjuvant regimen for HER2-positive early breast cancer (BC), the use of two HER2-directed agents is more effective in producing pathological complete (pCR) responses than trastuzumab alone. Nevertheless, highly effective dual anti-HER2 combination may be unnecessary in patients who already benefit from a single agent. We previously reported that our 41-gene TRAR score is an accurate predictor of response to trastuzumab, with low scores being predictive of response to trastuzumab and favorable prognosis (Triulzi T. et al., 2015). PATIENTS AND METHODS: Tissue specimens from HER2-positive BC patients of Neo-ALTTO trial who received neoadjuvant trastuzumab and/or lapatinib plus paclitaxel were included in this study. Analysing RNA from fresh tissue using the 41-gene signature test, the area under the ROC curve (AUC) and the corresponding 95% confidence interval was computed to evaluate the predictive ability of TRAR score with respect to pCR, the primary endpoint of Neo-ALTTO. The prognostic role of TRAR score was investigated using a Cox regression model in univariate fashion. The patterns of Event Free Survival (EFS) according to the dichotomized TRAR score were estimated using the Kaplan–Meier method. RESULTS: The TRAR score was assessed for 226 of the 455 (49.7%) patients enrolled in the Neo-ALTTO study: 136 (60%) presented with T2 tumors, 188 (83%) with N0/1 and 128 (56.6%) with estrogen receptor negative BC. In details, basal TRAR score was available for 69, 79 and 78 patients assigned to neoadjuvant trastuzumab, lapatinib, and their combination, respectively. Overall, patients achieving a pCR showed significantly lower levels of TRAR score than those with residual disease (p CONCLUSION: Overall, we show that our 41-gene signature is accurate in predicting patient response to neoadjuvant HER2 targeted therapy in terms of pCR. In particular, low levels of TRAR score can identify a HER2-positive breast cancer subgroup highly responsive to trastuzumab as monotherapy for whom combination with other HER2-targeted drugs does not appear justified and may be one tool used for exploring de-escalating strategies without sacrificing outcomes. Citation Format: Di Cosimo S, Triulzi T, De Cecco L, Pizzamiglio S, de Azambuja E, Fumagalli D, Pusztai L, Harbeck N, Izquierdo M, de la Pena L, Huober J, Baselga J, Piccart M, Verderio P, Tagliabue E. Identifying clinically relevant subgroups of women with HER2-positive breast cancer: An analysis of Neo-ALTTO using the 41-gene TRAR score [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-09-03.

BACKGROUND The new entity “diffuse-midline-glioma H3K27M-mutant”(DMG) was defined in the 2016 WHO classification. This tumor subtype, regardless of histological features, is associated with an aggressive clinical behavior and poor prognosis, sharing the same outcome as DIPG. MATERIAL AND METHODS We report our experience from 2016 to 2018 in treating patients with radiotherapy and concomitant Nimotuzumab/Vinorelbine, as already published for DIPG’s patients (DOI10.1007/s11060-014-1428-z). All patients’ parents provided written informed consent for treatment. RESULTS Patients were 9: 7/9 females, median age at diagnosis 13 years (range 1–26); 8 with localized disease, 1 with spinal fluid (CSF) dissemination. Five patients had thalamic disease (1 bithalamic), 2 ponto-cerebellar (one with 2 lesions), 1 pineal gland and one with disseminated CSF disease at diagnosis. 3/9 were biopsed in thalamus, the others had partial resection. Central neuropathological review with confirmed histopathological diagnosis of DMG mutated was necessary for inclusion. At the first pathological evaluation tumors had been classified according to the fourth edition of the WHO 2007 as glioblastoma (n=4), anaplastic astrocytoma grade III (n=1), diffuse astrocytoma grade II (n=1), and 1 glial tumor with oligodendroglial features; 2 patients already had DMG at the first pathological evaluation. Immunohistochemical analysis revealed p53 mutation in 3 patients. All 9 patients were evaluated on serial plasma samples with NGS targeted panel (Pan-Cancer) and 6/9 presented p53 mutation in at least one of the samples. 8/9 expressed MAP2K1 gain of function at liquid biopsy. The small number of patients did not allow correlations with the prognosis. Two patients completed the scheduled 2 years of treatment: one had a local relapse at 37 months after diagnosis, the other is alive without evidence of progression at 30 months. With a median follow up of 14.4 months, PFS and OS were 33% and 77% at 1 year; OS was 22% at 2 years, with 2 patients long survivors at 36 and 40 months after diagnosis. CONCLUSION The molecular and phenotypic pattern of DMG allows to include patients in clinical trials/registry shared with patients suffering from DIPG. Our knowledge is still limited about this entity; new insights into molecular profile should help us to study new drugs directed against the effects of the H3K27M mutations and to define new diagnostic/prognostic approach as liquid biopsy able to correlate treatments and prognosis

Objectives About 30% of Epithelial Ovarian Cancer (EOC) patients present with early-stage disease (FIGO stage I–II). However, in spite of the generally favourable prognosis, eEOC have heterogeneous risk of relapse, ranging from 13% to over 40% in various reports. We previously identified 35 miRNAs that predicted risk of progression or relapse in advanced EOC and used them to create a prognostic model, the 35-miRNA-based predictor of Risk of Ovarian Cancer Relapse or progression (MiROvaR). The aim of the current study is to test MiROvaR performance to predict risk of progression or relapse in patients with eEOC. Methods Patients with eEOC who underwent primary surgery at our Institution between 1994 and 2014 were included. The primary endpoint was progression-free survival. In particular, it was assessed the ability of MiROvaR to predict progression-free survival with Kaplan-Meier curves and the log-rank test. Results A total of 80 patients were included in the study with a median follow-up time of 68.2 months (CI: 61.3–80.7). MiROVaR classified 37 patients (46.3%) at high-risk (14 events, median PFS: 117 months, 95%CI: 62-nyr) and 43 patients (53.7%) at low-risk of relapse (2 events, median PFS time: nyr; 95%CI:nyr). Kaplan-Meier curves confirmed the significantly different PFS time (figure 1) for the two groups with HR=10.13 (95%CI: 2.3–4.4, p=0.00015) for the high-risk patients. Conclusions MiROvaR confirmed as a potential predictor of EOC progression and has prognostic value independent of relevant clinical covariates also in patients with eEOC. MiROvaR warrants further investigation for the development of a clinical-grade prognostic assay.

Background Circulating microRNAs (cmiRNAs) are emerging as promising non-invasive biomarkers in cancer for their diagnostic, prognostic and predictive potential. Several platforms have been developed to determine the presence of miRNAs in biological fluids, including specific focus panels. In this pilot study, we aimed at comparing different platforms for detection of cmiRNAs against the same cohort of plasma samples alongside RT-qPCR and NGS technologies. Methods RNA was isolated from 24 plasma samples obtained from lung cancer patients (n = 12, including one replicate) and healthy heavy smokers (n = 12, including two replicates). Six different platforms were used to detect human cmiRNAs. Five of them were high-throughput technologies, three RT-qPCR-based (TaqMan OpenArray and TaqMan OpenArray Advanced MicroRNA Panels by ThermoFisher; miRCURY LNA miRNA miRNome PCR panel by Qiagen) and two NGS-based (EdgeSeq miRNA byHTG and QiaSeq miRNA by Qiagen). The sixth platform, RT-qPCR-based and serum/plasma focused, was TaqMan Advanced miRNA Human Card by ThermoFisher. Samples were handled according to manufacturer’s instructions and data from each platform were analyzed using specific recommended thresholds. Results Selecting cmiRNAs yielding signals above background, we highlighted a subset commonly detected by all platforms. Strong concordance and correlation coefficients (>0.8) between the three replicate samples were obtained in most platforms, from independent RNA extractions. For each sample, the number of cmiRNAs detected ranged from 90 to 140. We found that inter-platform correlations were highest for platforms based on similar chemistry and assay design. Between lung cancer patients and healthy donors, few deregulated miRNAs could be however commonly detected by all platforms and studies are ongoing to elucidate their role. Conclusions This study is one of the first comparing RT-qPCR, NGS-based high-throughput and serum/plasma focused platforms for human cmiRNA biomarker detection. Advantages and limits of all platforms and data on intra- and inter- reproducibility will be presented to help investigators in identifying technologies better suited for cmiRNAs screening or validation in clinical cohorts. Legal entity responsible for the study The authors. Funding Italian Association for Cancer Research (AIRC, Special Programme "5 X 1000", ED No12162) to GS and MS and by Italian Ministry of Health (funds obtained through an Italian law that allows tax-payers to allocate the “5 × 1000” share of their payments to research) to LDC. Disclosure All authors have declared no conflicts of interest.

Table S6. Process networks obtained with Metacore analyzing only down-regulated genes. (DOCX 15 kb)

Background Effective target therapies for intrahepatic cholangiocarcinoma (ICC) have not been identified so far. One of the reasons may be the genetic evolution from primary (PR) to recurrent (REC) tumors. We aim to identify peculiar characteristics and to select potential targets specific for recurrent tumors. Eighteen ICC paired PR and REC tumors were collected from 5 Italian Centers. Eleven pairs were analyzed for gene expression profiling and 16 for mutational status of IDH1. For one pair, deep mutational analysis by Next Generation Sequencing was also carried out. An independent cohort of patients was used for validation. Results Two class-paired comparison yielded 315 differentially expressed genes between REC and PR tumors. Up-regulated genes in RECs are involved in RNA/DNA processing, cell cycle, epithelial to mesenchymal transition (EMT), resistance to apoptosis, and cytoskeleton remodeling. Down-regulated genes participate to epithelial cell differentiation, proteolysis, apoptotic, immune response, and inflammatory processes. A 24 gene signature is able to discriminate RECs from PRs in an independent cohort; FANCG is statistically associated with survival in the chol-TCGA dataset. IDH1 was mutated in the RECs of five patients; 4 of them displayed the mutation only in RECs. Deep sequencing performed in one patient confirmed the IDH1 mutation in REC. Conclusions RECs are enriched for genes involved in EMT, resistance to apoptosis, and cytoskeleton remodeling. Key players of these pathways might be considered druggable targets in RECs. IDH1 is mutated in 30% of RECs, becoming both a marker of progression and a target for therapy.

Table S4. Process networks obtained with Metacore analyzing only up-regulated genes. (DOCX 15 kb)

Table S3. Pathway maps obtained with Metacore analyzing only up-regulated genes. (DOCX 16 kb)

Table S1. Clinical pathological characteristics of ICC patients. (DOCX 19 kb)

Background To investigate the activity and safety of afatinib in the preoperative treatment of squamous cell carcinoma of the head and neck (SCCHN). Patients and methods This study was an open-label, randomized, multicenter, phase II window of opportunity trial. Treatment-naive SCCHN patients selected for primary curative surgery were randomized (5 : 1 ratio) to receive afatinib during 14 days (day –15 until day –1) before surgery (day 0) or no treatment. Tumor biopsies, 2-[fluorine-18]-fluoro-2-deoxy- d -glucose positron emission tomography (FDG-PET), and magnetic resonance imaging (MRI) were carried out at diagnosis and just before surgery. The primary end point was metabolic FDG-PET response (according to EORTC guidelines). Other end points included response assessment based on the Response Evaluation Criteria In Solid Tumors (RECIST) v1.1, dynamic contrast-enhanced (DCE)-MRI, diffusion weighted (DW)-MRI, safety, and translational research (TR). Results Thirty patients were randomized: 25 to afatinib and 5 to control arm. Of the 23 eligible patients randomized to afatinib, 16 (70%; 95% CI: 47% to 87%) patients had a partial metabolic FDG-PET response (PMR). Five patients (22%; 95% CI: 8% to 44%) showed a partial response by RECISTv1.1. Responses assessed via DCE-MRI and DWI-MRI did not show a strong association with PMR or RECIST. One patient discontinued afatinib after 11 days for grade 3 diarrhea with subsequent renal failure and 24 days delay in surgery. No grade 4 toxicities or surgical comorbidities related to afatinib were reported. TR results indicated that PMR was more frequent in the tumors with high Cluster3-hypoxia score expression and with TP53 wild type. Conclusion Afatinib given for 2 weeks to newly diagnosed SCCHN patients induces a high rate of FDG-PET partial metabolic response and partial response according to RECISTv1.1. Afatinib can be safely administered before surgery. Although exploratory, the hypoxic gene signature needs further investigations as a predictive biomarker of afatinib activity. Clinical trial registration ClinicalTrials.gov: NCT01538381

Table S5. Pathway maps obtained with Metacore analyzing only down-regulated genes. (DOCX 15 kb)

Trastuzumab, a recombinant humanized monoclonal antibody directed to the HER2 protein, has shown survival benefits in women with HER2-positive breast cancer, and treatment is now FDA-approved in combination with chemotherapy. Nonetheless, some patients relapse after treatment, underscoring the need to identify patients for whom chemotherapy + trastuzumab is adequate versus patients requiring additional drugs. To search for factors predictive of relapse in HER2-positive breast carcinoma patients treated adjuvantly with trastuzumab, we conducted gene expression profiling analysis in 53 cases selected among a cohort of 243 patients (32 of whom relapsed) treated in the clinic with chemotherapy + trastuzumab in our institute during 2006-2009, with median follow-up of 32 months; the 53 cases comprised 23 of the relapsed and 30 of the non-relapsed patients with similar clinico-pathological characteristics (size, lymph node involvement and estrogen receptor positivity) for whom sufficient material was available. RNA extracted from formalin-fixed, paraffin-embedded (FFPE) was profiled using HumanHT12_v4 wgDASL expression BeadChips (Illumina). A Cox's proportional hazard model was used to estimate the association between gene expression and relapse-free survival (RFS), and a multivariate permutation test was used to check the proportion of false discoveries. Analysis identified 330 probes corresponding to 308 unique genes as significantly associated to RFS (a We also tested the feasibility of developing a predictive model including the gene expression data. Based on 10-fold cross-validation, our model was able to stratify patients into two groups (high and low risk), with a 4-year RFS probability of 14.5% in high-risk versus 87% in low-risk tumors (HR = 8.33, 95% CI = 3.53-18.18, p Together, our results suggest the usefulness of molecular characteristics of primary tumors as indicators of early relapse in patients treated adjuvantly with trastuzumab and as tools to identify patients for whom additional treatments are required. Supported by AIRC. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-12-03.

Background: Aberrant choline metabolism has been proposed as a novel cancer hallmark. We recently showed that epithelial ovarian cancer (EOC) possesses an altered MRS-choline profile, characterised by increased phosphocholine (PCho) content to which mainly contribute over-expression and activation of choline kinase-alpha (ChoK-alpha). Methods: To assess its biological relevance, ChoK-alpha expression was downmodulated by transient RNA interference in EOC in vitro models. Gene expression profiling by microarray analysis and functional analysis was performed to identify the pathway/functions perturbed in ChoK-alpha-silenced cells, then validated by in vitro experiments. Results: In silenced cells, compared with control, we observed: (I) a significant reduction of both CHKA transcript and ChoK-alpha protein expression; (II) a dramatic, proportional drop in PCho content ranging from 60 to 71%, as revealed by 1H-magnetic spectroscopy analysis; (III) a 35–36% of cell growth inhibition, with no evidences of apoptosis or modification of the main cellular survival signalling pathways; (IV) 476 differentially expressed genes, including genes related to lipid metabolism. Ingenuity pathway analysis identified cellular functions related to cell death and cellular proliferation and movement as the most perturbed. Accordingly, CHKA-silenced cells displayed a significant delay in wound repair, a reduced migration and invasion capability were also observed. Furthermore, although CHKA silencing did not directly induce cell death, a significant increase of sensitivity to platinum, paclitaxel and doxorubicin was observed even in a drug-resistant context. Conclusion: We showed for the first time in EOC that CHKA downregulation significantly decreased the aggressive EOC cell behaviour also affecting cells' sensitivity to drug treatment. These observations open the way to further analysis for ChoK-alpha validation as a new EOC therapeutic target to be used alone or in combination with conventional drugs.

This study presents a first-level spatial assessment of the susceptibility to earthquake-induced landslides in the seismic area of the Agri Valley (Basilicata Region, southern Italy), which hosts the largest onshore oilfield and oil/gas extraction and pre-treatment plant in Europe and is the starting point of the 136-km-long pipeline that transports the plant’s products to the refinery located in Taranto, on the Ionian seacoast. Two methodologies derived from the ones proposed by Newmark (Geotechnique 15(2):139–159, 1965) and Rapolla et al. (Eng Geol 114:10–25, 2010, Nat Hazards 61:115–126, 2012. doi: 10.1007/s11069-011-9790-z ), based on different modelling approaches, were implemented using the available geographic information system tools, which allowed a very effective exploitation of the two models capability for regional zoning of the earthquake-induced landslide hazard. Subsequently, the results obtained from the two models were compared by both visual evaluation of thematic products and statistical correlation analysis of quantitative indices, such as the Safety Index based on the Newmark’s approach and the Susceptibility Index from Rapolla’s model. The comparison showed a general agreement in highlighting the most critical areas. However, some slight differences between the two models’ results were observed, especially where rock materials and steep slopes are prevailing.

Sensitive tumor biomarkers able to monitor disease progression would contribute to post-surgical treatment decision making in early breast cancer. We investigated the feasibility of using circulating tumor DNA (ctDNA) to early detect new disease manifestations in serial plasma samples collected during post-surgery follow-up from patients operated for stage I breast cancer from 1992 to 1993 at Istituto Nazionale Tumori in Milan. Forty patients that underwent radical or conservative surgery for T1/T2-N0-M0 breast cancer and that were followed for at least 15 years were included in a pilot study for the retrospective analysis of ctDNA on at least 3 plasma samples obtained during follow-up. To assess the feasibility of ctDNA analysis in archival plasma samples collected in heparin and stored from 10 to 25 years, preliminary experiments demonstrated that ctDNA was not affected by: 1) heparinase I digestion of extracted DNA and 2) DNA pre-amplification step to overcome limitations due to small plasma aliquots. Mutational analysis of breast cancer tissues was performed by Ion Torrent-targeted next generation sequencing and the identified Single Nucleotide Variations (SNV) were first validated and then tracked in plasma samples by using ad hoc digital polymerase chain reaction assays. One or more SNVs were identified in tumor tissue specimens and validated in 27/40 cases. Among those 27 breast cancers, 6 cases relapsed locally, 4 in distant sites, and 17 remained disease-free for the entire follow-up. ctDNA was undetectable during the post-surgical follow-up in 16/17 disease-free women up to 160 months of surgery, while it was detectable in 9/10 patients developing unfavorable events and anticipated the clinical diagnosis of relapse in 7/10 patients with a median lead time of 20 months. Our results are the first to associate mutation tracking to local recurrence and indicate that in patients with early breast cancer ctDNA monitoring during post-operative follow-up can anticipate the diagnosis of new disease manifestations, thus potentially allowing prompt treatments. These findings establish the rational to plan prospective studies to evaluate in the early breast cancer context the potential of ctDNA as a non-invasive and sensitive biomarker for monitoring tumor progression. Based on these results, we activated in 2016 a prospective observational study to confirm the predictive value of ctDNA on local and distant relapse in patients with early and localized triple negative breast cancer. As for May 2017, 145 patients with triple negative tumors were potentially enrolled for a ctDNA-based post-surgical follow-up: 111 cases at first diagnosis and 34 cases at surgery after neo-adjuvant treatment. One hundred-ten women accepted to participate in the study and signed a specific informed consent, whereas 22 patients refused to participate and 13 were lost to follow-up. For 94 patients (66 at initial diagnosis and 28 after neoadjuvant chemotherapy) plasma samples have been already longitudinally collected and DNA sequencing is currently in progress. Citation Format: Daidone MG, Di Cosimo S, Veneroni S, Cascone F, De Cecco L, Dugo M, Folli S, Bianchi GV, Tamborini E, Busico A, Appierto V. Circulating tumor DNA detection anticipates disease recurrence in early stage breast cancer: A pilot study generating an observational confirmatory trial [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-02-19.

Background: gene expression profiling has distinguished sporadic breast tumour classes with genetic and clinical differences. Less is known about the molecular classification of familial breast tumours, which are generally considered to be less heterogeneous. Here, we describe molecular signatures that define BRCA1 subclasses depending on the expression of the gene encoding for oestrogen receptor, ESR1. Methods: for this purpose, we have used the Oncochip v2, a cancer-related cDNA microarray to analyze 14 BRCA1-associated breast tumours. Results: signatures were found to be molecularly associated with different biological processes and transcriptional regulatory programs. The signature of ESR1-positive tumours was mainly linked to cell proliferation and regulated by ER, whereas the signature of ESR1-negative tumours was mainly linked to the immune response and possibly regulated by transcription factors of the REL/NFκB family. These signatures were then verified in an independent series of familial and sporadic breast tumours, which revealed a possible prognostic value for each subclass. Over-expression of immune response genes seems to be a common feature of ER-negative sporadic and familial breast cancer and may be associated with good prognosis. Interestingly, the ESR1-negative tumours were substratified into two groups presenting slight differences in the magnitude of the expression of immune response transcripts and REL/NFκB transcription factors, which could be dependent on the type of BRCA1 germline mutation. Conclusion: this study reveals the molecular complexity of BRCA1 breast tumours, which are found to display similarities to sporadic tumours, and suggests possible prognostic implications.

We describe a microarray experiment using the MCF-7 breast cancer cell line in two different experimental conditions for which the same number of independent pools as the number of individual samples was hybridized on Affymetrix GeneChips. Unexpectedly, when using individual samples, the number of probe sets found to be differentially expressed between treated and untreated cells was about three times greater than that found using pools. These findings indicate that pooling samples in microarray experiments where the biological variability is expected to be small might not be helpful and could even decrease one's ability to identify differentially expressed genes.

One of the main problems in managing ranges used for extensive pastoralism is the difficulty of obtaining reliable estimates of grass biomass over very large areas. Estimates of grass biomass are useful as an indicator of both available forage and risk of soil erosion. Nevertheless, large scale field measurements are expensive and time-consuming. The use of satellite images may provide a complementary means of estimating grass biomass over very large areas at a reasonable cost. The aim of this study was to test the use of Landsat satellite data for estimating grass biomass in a mountainous range in central Italy used primarily for sheep breeding. During each of four ground campaigns carried out over two years, grass was cut and its biomass measured in 60-90 test plots. Four Landsat images taken simultaneously to the ground campaigns were processed to obtain several vegetation indexes calculated for each ground test plot. The vegetation indexes showed significant correlations with measured grass biomass. The Normalized Difference Vegetation Index (NDVI) provided the most accurate estimate of grass biomass. When data for each of the four ground campaigns were analyzed separately, correlations for early summer campaigns were higher than correlations for late summer campaigns, indicating that when the ratio of dry/green biomass increases, satellite estimate becomes less accurate. Overall, our results show that satellite data can provide a useful source of biomass information for the management of large ranges.

Genomics adds a new dimension to genetic analysis, shifting the focus from the study of a single gene to the whole genome. We have successfully applied the genomics approach based on microarray to the study of genes involved in barley responses to cold stress. About 900 EST clones from barley were obtained from a cDNA library of cold acclimatized leaves of cv. Nure and arrayed, and gene expression analysis done on cold acclimatized vs. control plants. The system allowed for reliable detection of differences in mRNA expression levels, and was confirmed by the finding that numerous previously reported cold-related genes were differentially expressed in treated and untreated plants when evaluated in our arrays. The expression profiles of a sample of genes analysed by the array were confirmed by quantitative RT-PCR. Previously, identification of novel plant genes was achieved considering a few genes at a time; now many genes can be found as up- or down-regulated based on a one step procedure. Many of the genes we found to be up- or down-regulated do not have an assigned function. This includes 15 of the 78 up-regulated and 8 of the 45 down-regulated clones. Our results add new genes to the group of cold-regulated genes and provide the opportunity to better understand the complex mechanism of stress tolerance.