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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.

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.

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.

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

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.

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.

Heterogeneous expression of the collagen receptor DDR1 in chronic lymphocytic leukaemia and correlation with progression

Background Head and neck squamous cell carcinoma refers to a heterogeneous disease frequently aggressive in its biologic behavior. Despite the improvements in the therapeutic modalities, the long-term survival rate remained unchanged over the past decade and patients with this type of cancer are at a high risk of developing recurrence. For this reason, there is a great need to find better ways to foresee outcome, to improve treatment choices, and to enable a more personalized approach. Patients and methods Nine microarray gene expression datasets, reporting survival data of a total of 841 samples, were retrieved from publicly repositories. Three datasets, profiled on the same version of microarray chips, were selected and merged following a meta-analysis approach to build a training set. The remaining six studies were used as independent validation sets. Results The training set led us to identify a 172-gene signature able to stratify patients in low or high risk of relapse [log-rank, P = 2.44e-05; hazard ratio (HR) = 2.44, 95% confidence interval (CI) 1.58–3.76]. The model based on the 172 genes was validated on the six independent datasets. The performance of the model was challenged against other proposed prognostic signatures (radiosensitivity index, 13-gene oral squamous cell carcinoma signature, hypoxia metagene, 42-gene high-risk signature) and was compared with a human papillomavirus (HPV) signature: our model resulted independent and even better in prediction. Conclusions We have identified and validated a prognostic model based on the expression of 172 genes, independent from HPV status and able to improve assessment of patient's risk of relapse compared with other molecular signatures. In order to transpose our model into a useful clinical grade assay, additional work is needed following the framework established by the Institute of Medicine and REMARK guidelines.

Efficacy of medical treatment for the management of endometriosis has been documented in several trials, but clinical results cannot always be maintained after the suspension of treatment. Surgical treatment, either laparotomic or laparoscopic, is affected by up to 20% in the recurrence of clinical symptoms after long-term follow-up. The appearance of endometriosis is heterogeneous, its functional status is variable and could lack hormone responsiveness. After medical, surgical or combined treatment the persistence of the failure of defence mechanisms accounts for the recurrence of disease. Unfortunately, all schemes to classify stages of endometriosis have so far failed to identify manifestations of the disease that respond in a predictable way to specific treatments. An analysis of the morphological appearance, implant biological activity and immune system involvement might better define the roles for medical management of endometriosis.

A double-blind clinical trial was performed in two clinical centers to evaluate the efficacy and the safety of two dose schedules of fenticonazole (one 600-mg or one 1000-mg ovule applied for 2 consecutive days) versus placebo in the treatment of vaginal trichomoniasis. Sixty-one patients were included in the study in the following three treatment groups: 600-mg ovules, 21 patients; 1000-mg ovules, 20 patients; and placebo, 20 patients. A vaginal swab was obtained before treatment and on day 7 for phase-contrast direct microscopic identification of Trichomonas species and a cultural examination was performed to exclude a possible mycotic or bacterial etiology. The severity of vaginal signs and symptoms (erythema, itching, discharge, edema, and burning) were evaluated with a semiquantitative scale (0 to 3). At baseline, all patients were positive for Trichomonas vaginalis on microscopic examination; on final evaluation the test was negative in 13 (65.0%) patients in the 600-mg group (one patient did not have a final examination performed), in 10 (58.8%) of the 1000-mg group, and in 3 (15.0%) of the placebo group. The difference between patients receiving active drug and placebo was highly significant (chi-square test, P ≤ 0.005), while no significant difference existed between the two groups of drug-treated patients. A highly significant reduction of the symptomatic scores was observed in both active-treatment groups as compared with placebo, but, as in the case of the microbiologic findings, no difference was found between the two groups of fenticonazole-treated patients. Side effects of mild or moderate intensity were present in three patients in the 1000-mg dose group. Two patients had a burning sensation and one had a burning sensation and discharge. One of the patients with a burning sensation interrupted the treatment. Such symptoms could also be part of the underlying disease.

Influence on the immune system activity by sex hormones has been widely reported. Fertile women are proner to the onset of autoimmune diseases than men, but this increased susceptibility disappears after menopause. The hormonal changes are very likely to be responsible for this event, but precise correlations between sex hormone levels and immune functions have not been defined. For this reason we have analyzed phenotype and natural cytotoxicity of peripheral blood lymphocytes (PBL) from 35 women in menopause, comparing them with the same parameters of 28 fertile and 8 postmenopausal women and correlating them with the hormonal pattern of each group. We have also considered 8 women with premature menopause. Hormonal levels have been detected by radioimmune assays, while PBL phenotype has been studied by immunofluorescence and FACS analysis. The natural killer (NK) cell activity has been calculated on the basis of a chromium release assay. Postmenopausal women showed a reduction of the number of total lymphocytes (1650 +/- 215 cells/mmc) in comparison to fertile women (2081 +/- 200 cells/mmc, P < 0.01). The decrease mainly involved B and CD4+ T lymphocyte subpopulations (P < 0.05 and P < 0.01, respectively). Women with premature menopause had lower percentage of CD4 lymphocytes (34% vs 47%, P < 0.01) and higher percentage of CD8 (30% vs 22%, P < 0.02) and NK cells (32% vs 14%, P < 0.009) than fertile women of the same age. The percentage of circulating lymphocytes expressing HLA class II antigens also resulted as being increased (22% vs 9%, P < 0.01). The number of total, CD2, CD4 T lymphocytes, B and NK cells correlated positively with LH and negatively with FSH serum levels (P < 0.05 and P < 0.002, respectively). PRL positively influenced CD2, CD4 and B lymphocyte numbers (P < 0.001). FSH and 17 beta-estradiol inversely affected CD8 and B lymphocyte numbers (P < 0.005 and P < 0.02, respectively). In conclusion, the increase of FSH and the decrease of PRL levels appear to be involved in the reduction of B and CD4 T lymphocytes thus lowering the risk for the onset of autoimmune diseases during and after menopause. Generalized activation of the immune system (raised expression of HLA class II antigens) with elevated numbers of cytotoxic subpopulations (CD8 and NK lymphocytes) is present in women affected by premature menopause suggesting the involvement of autoimmune dysregulation in the pathogenesis of this syndrome.

Fenretinide ( 4-HPR) is a synthetic retinoid with antitumor activity, which induces apoptosis in cancer cell lines of different histotypes. To identify genes contributing to its apoptotic activity in ovarian cancer cells, we monitored, by cDNA arrays, gene expression changes after 4-HPR exposure in A2780, a human ovarian carcinoma cell line sensitive to the retinoid. Among the differentially expressed transcripts, PLAcental Bone morphogenetic protein ( PLAB), a proapoptotic gene, was the most highly induced. In a panel of ovarian carcinoma cell lines with different 4-HPR sensitivities, PLAB upregulation was associated with cellular response to 4-HPR, its overexpression increased basal apoptosis and its silencing by small interfering RNA decreased the ability of 4-HPR to induce apoptosis. PLAB induction by 4-HPR was p53- and EGR-1 independent and was regulated, at least in part, by increased stability of PLAB mRNA. PLAB up-modulation by 4-HPR also occurred in vivo: in ascitic cells collected from patients with ovarian cancer before and after 4-HPR treatment, PLAB was upmodulated in 2/4 patients. Our results in certain ovarian cancer cell lines indicate a role for PLAB as a mediator of 4-HPR-induced apoptosis. The correlation of increased PLAB in vivo with antitumor activity remains to be established.

The mouse parathyroid hormone-like hormone (Pthlh) gene encodes three allelic variants characterized by amino acid substitutions that are associated with susceptibility (Pthlh(Pro)) or resistance (Pthlh(Thr) and Pthlh(SerAspTyr)) to two-stage skin carcinogenesis and to modulation of cell migration in vitro in transfected human cancer cells. cDNA microarray hybridization analysis of 8473 transcript clones revealed a similar gene expression profile for the Pthlh(Thr) and Pthlh(SerAspTyr) alleles but a distinct pattern for the Pthlh(Pro) allele, suggesting an association between a specific gene expression profile and biological function of the Pthlh alleles. Some of the genes modulated by the Pthlh alleles, e.g., ANXA1, CCL2, FN1 and TFF3, play a role in cell migration and may represent candidate targets for this Pthlh function. Our study demonstrates the potential usefulness of gene expression profiling of genetic variants for the functional characterization of candidate cancer modifier genes.

Aims : To describe a tumour with morphological and immunophenotypic characteristics of epithelioid variant of pleomorphic liposarcoma. Pleomorphic liposarcoma is a very rare variant of liposarcoma defined morphologically by the presence of pleomorphic lipoblasts showing peculiar epithelial-like features that can be confused with primary or metastatic carcinoma. Methods and results : Molecular analysis demonstrated for the first time the presence of FUS-CHOP transcript in this liposarcoma variant. Microarray analysis revealed a gene expression profile related to a more aggressive tumour type when compared with other myxoid/round cell liposarcomas. Conclusions : The present data show that the epithelioid variant of pleomorphic liposarcoma represents a further variant of myxoid liposarcoma sharing the FUS-CHOP fusion transcript but carrying a distinct expression profile, in keeping with its aggressive clinical course.

20 nonpregnant patients with chronic vaginal candidosis were divided randomly into 5 groups of 4 patients. They all received itraconazole 200mg once daily for 3 consecutive days. A blood sample and vaginal biopsy were obtained 1 hour after each administration as well as 25 and 50 hours after the last drug intake. The results show that the itraconazole levels in the vaginal mucosa are comparable with the corresponding blood levels, but that after the end of treatment, drug levels in the vagina remain elevated for much longer than the corresponding blood levels. These data suggest that a regimen of itraconazole 200mg once daily for 3 days results in therapeutic tissue levels at the site of infection for more than 5 days.

Aim: Clinical and radiological diagnostics are not able to predict nodal or extranodal extension known to negatively impact on disease outcome in oral cavity squamocellular cancer (OCSCC). We assessed whether a gene-expression signature from specimens obtained from primary tumour could predict for nodal status. Methods: A series of archival specimens from patients with stage III-IV OCSCC treated with surgery at T and N level as first treatment from 1989 to 2008 was collected. The histological samples were randomly chosen in order to have an equal number of cases with pathological negative nodes (pN0), positive nodes without extracapsular extension (pN + ECS-) and positive nodes with extracapsular extension (pN + ECS+). The histological specimens of primary disease were microdissected in order to obtain one sample from the central area of the tumour (pTcent) and one from the peripheral area (pTperiph). The samples of primary tumour were profiled for gene expression on DASLwg Illumina BeadChips. Results: We present hereafter the results of the first 29 patients in the trial (pN0 = 10; pN + ECS- = 9; pN + ECS + = 10). Gene expression profile resulted in a data matrix containing about 18600 detected genes. We focused our attention on the genes differentially expressed between pN0 and pN+ and between pN + ECS- and pN + ECS+. Imposing a significance threshold of false discovery rate (FDR) Conclusions: The research revealed different gene expression signatures on primary tumour useful to predict nodal status in OCSCC. Mature results of the analysis on a higher number of cases will be presented during the meeting. *Acknowledgment: Italian Ministry of Health call “Ricerca Finalizzata” (Project code: GR -2009 -1492184). Disclosure: All authors have declared no conflicts of interest.