Circulating cell free DNA and citrullinated histone H3 as useful biomarkers of NETosis in endometrial cancer.

BACKGROUND: Cancer mortality is mainly caused by organ failure and thrombotic events. It has been demonstrated that NETosis, a chromatin release mechanism implemented by neutrophils, may contribute to these lethal systemic effects. Our aim was to investigate NETosis biomarkers in endometrial cancer (EC). METHODS: The experiments were conducted on 21 healthy subjects (HS) with no gynecological conditions, and on 63 EC patients. To assess the presence of NETosis features, IHC and IF was performed using antibodies against citrullinated histone H3 (citH3), neutrophil elastase (NE) and histone 2B. Serum levels of cell free DNA (cfDNA), cell free mitochondrial DNA (cfmtDNA) and citH3 were measured by qPCR using one microliter of deactivated serum, and by ELISA assay respectively. Fragmentation pattern of serum cfDNA was analyzed using the Agilent 2100 Bioanalyzer and High Sensitivity DNA Chips. Receiver operating characteristic (ROC) analysis was used to identify a cut off for cfDNA and cfmtDNA values able to discriminate between ECs and HSs. Correlation analysis and multiple correspondence analysis (MCA) between cfDNA, mtcfDNA, citH3 and blood parameters were used to identify the potential association among serum parameters in EC grades. RESULTS: We demonstrated the presence of NETosis features in tissues from all EC grades. Serum cfDNA and cfmtDNA levels discriminate ECs from HSs and a direct correlation between citH3 and cfDNA content and an inverse correlation between cfmtDNA and citH3 in EC sera was observed, not detectable in HSs. MCA indicates cfDNA, cfmtDNA and citH3 as features associated to G1 and G2 grades. A correlation between increased levels of cfDNA, citH3 and inflammation features was found. Finally, serum nucleosomal cfDNA fragmentation pattern varies in EC sera and correlates with increased levels of cfDNA, citH3, lymphocytes and fibrinogen. CONCLUSION: Our data highlight the occurrence of NETosis in EC and indicate serum cfDNA and citH3 as noninvasive biomarkers of tumor-induced systemic effects in endometrial cancer.

Che-1/AATF-induced transcriptionally active chromatin promotes cell proliferation in multiple myeloma.

Multiple myeloma (MM) is a hematologic malignancy produced by a clonal expansion of plasma cells and characterized by abnormal production and secretion of monoclonal antibodies. This pathology exhibits an enormous heterogeneity resulting not only from genetic alterations but also from several epigenetic dysregulations. Here we provide evidence that Che-1/AATF (Che-1), an interactor of RNA polymerase II, promotes MM proliferation by affecting chromatin structure and sustaining global gene expression. We found that Che-1 depletion leads to a reduction of "active chromatin" by inducing a global decrease of histone acetylation. In this context, Che-1 directly interacts with histones and displaces histone deacetylase class I members from them. Strikingly, transgenic mice expressing human Che-1 in plasma cells develop MM with clinical features resembling those observed in the human disease. Finally, Che-1 downregulation decreases BRD4 chromatin accumulation to further sensitize MM cells to bromodomain and external domain inhibitors. These findings identify Che-1 as a promising target for MM therapy, alone or in combination with bromodomain and external domain inhibitors.

Iodixanol versus iopromide in cancer patients: Evidence from a randomized clinical trial.

To assess the safety profile of iso-osmolar contrast medium (CM) versus low osmolar CM in cancer patients with an estimated glomerular filtration rate (eGFR) >60 ml/min. In this multicenter, blind trial of patients seeking a chest-abdomen-pelvis contrast enhanced computed tomography (CT) with iodated CM, participants were centrally randomized to iodixanol or iopromide. Contrast induced nephropathy (CIN) at 24 and/or 72 hr were our primary outcomes. We further considered irreversible CIN, average eGFR percentage variation (%Δ), and adverse events (AEs). Overall, 607 patients were enrolled. Among them, 497 eligible patients were randomized to iodixanol (N: 247) or iopromide (N: 250). No differences emerged by descriptive characteristics. Seven and 3 CIN at 24 hr (p = 0.34) and 8 and 2 CIN at 72 hr (p = 0.11) occurred in the iopromide and iodixanol group, respectively. Within the subgroup of individual patients who developed CIN (N: 17), the event rate was higher in the iopromide arm (p = 0.045). No cases of permanent CIN or significant differences in terms of AEs or GFR %Δ were observed. Our results suggest a more favorable safety profile of iodixanol versus iopromide. Adequately sized trials with similar design are warranted to confirm our findings and clarify the underlying biological mechanisms.

Circulating cell-free DNA content as blood based biomarker in endometrial cancer.

BACKGROUND: Altered circulating cell-free DNA (cfDNA) levels are related to cancer development and aggressiveness. Up to now, very few studies have been performed for evaluating cfDNA content in endometrial cancer (EC). METHODS: First, we measured cfDNA release in blood serum of EC cancer patients collected before surgery and before the beginning of any treatment by SYBR Gold assay and correlated it with tumor aggressiveness. We also assessed the relative mitochondrial cell-free DNA (cfmtDNA) content by qRT-PCR. Next, we correlated cfDNA levels with BMI, age, hypertension and inflammation markers. RESULTS: CfDNA levels are higher in G2 and G3 compared with G1 EC sera. A significant modulation of cfDNA content was detected in sera from patients with BMI>30 compared with those with BMI<30. We observed a further and significant alteration in cfDNA level in hypertensive patients with G2-G3, but not in G1 EC. Analysis of preoperative neutrophil-to-lymphocyte (NLR) and monocyte-to-lymphocyte (MLR) ratios suggests a contribution of the host response in the altered cfDNA levels in EC. CONCLUSIONS: Our data indicate that assessment of total and mitochondrial cfDNA levels in blood sera and the relative NLR and MLR in blood obtained from preoperative patients may help clinical management and prognosis in EC.

Temozolomide low-dose chemotherapy in newly diagnosed low-grade gliomas: activity, safety, and long-term follow-up.

PURPOSE: To explore the efficacy and toxicity of an extended schedule of temozolomide (50 mg/mq 1 week on/1 week off) in a population of newly diagnosed low-grade gliomas (LGG). METHODS: Primary endpoints were progression-free survival (PFS) at 12 and 24 months and response rate evaluated with Response Assessment in Neuro-Oncology Criteria. Secondary endpoints were clinical benefit (reduction of seizures frequency), reduction of steroid, and modifications of Karnofsky Performance Status. RESULTS: From 2006 to 2009, we enrolled 14 consecutive patients with newly diagnosed LGG: 8 grade II astrocytomas, 2 oligodendroglioma, and 4 oligo-astrocytoma. Temozolomide was administered for 18 cycles (mean) per patient (range 3-24 cycles). In 57.5% (n = 8), we observed stable disease, 28.5% (n = 4) presented a minor response, and 14% (n = 2) showed progression. Five patients presented early progression during the first year of treatment and the study was stopped. A relevant clinical benefit was observed in 85% of patients (seizure control). After 6 years of follow-up, only 4 patients died. Prolonged PFS was associated with 1p-19q codeletion over 1p-19q intact (35 vs 4 months; p<0.04) and IDH1 mutation over IDH1 wild-type (36 vs 6 months; p<0.009). CONCLUSIONS: The study was interrupted for the high rate of progression observed in the first 14 patients enrolled. However, our results show that an extended low dose of temozolomide presents interesting activity with objective response and clinical benefit, but does not seem to prevent progression in patients presenting unfavorable molecular prognostic factors.

Flow cytometry remission by Ig light chains ratio is a powerful marker of outcome in multiple myeloma after tandem autologous transplant: a real-life study.

BACKGROUND: The achievement of complete response (CR) significantly correlates with a better clinical outcome in multiple myeloma (MM) patients treated with autologous stem cell transplant (ASCT). The depth of response is one of the most relevant factors to predict patient's outcome, however the definition of CR through standard criteria has shown several limitations. METHODS: In this study we evaluated the minimal residual disease (MRD) in 50 consecutive MM patients who underwent an up-front tandem ASCT in our center, using a single-tube six-colors flow cytometry assay (FC) based on intra-cytoplasmic immunoglobulin (cy-Ig) light chains ratio evaluated on patient-specific plasma cells (PC) immune profile, in a real-life setting. RESULTS: With a sensitivity up to 10(-5), clonal-PC were documented by FC in 36.4% (12/33) of patients in conventional CR after second transplant. The number of flow MRD-negative patients significantly increased after induction and first ASCT, but not between first and second transplant. The 5-years progression-free survival (5ys-PFS) of flow MRD-negative patients after second transplant was significantly better than patients who remained MRD-positive considering both all patients (5ys-PFS: 70% vs 5%) and patients in CR according to standard criteria (5ys-PFS: 67% vs 0%). CONCLUSIONS: FC remission through cy-Ig light ratio on PC sub-populations is a sensitive, highly informative, low-cost and routinely applicable MRD assay, a powerful tool in treatment response evaluation and a crucial marker of outcome in MM.

PCA3 in prostate cancer and tumor aggressiveness detection on 407 high-risk patients: a National Cancer Institute experience.

BACKGROUND: Prostate cancer (PCa) is the most common male cancer in Europe and the US. The early diagnosis relies on prostate specific antigen (PSA) serum test, even if it showed clear limits. Among the new tests currently under study, one of the most promising is the prostate cancer gene 3 (PCA3), a non-coding mRNA whose level increases up to 100 times in PCa tissues when compared to normal tissues. With the present study we contribute to the validation of the clinical utility of the PCA3 test and to the evaluation of its prognostic potential. METHODS: 407 Italian men, with two or more PCa risk factors and at least a previous negative biopsy, entering the Urology Unit of Regina Elena National Cancer Institute, were tested for PCA3, total PSA (tPSA) and free PSA (fPSA and f/tPSA) tests. Out of the 407 men enrolled, 195 were positive for PCa and 114 of them received an accurate staging with evaluation of the Gleason score (Gs). Then, the PCA3 score was correlated to biopsy outcome, and the diagnostic and prognostic utility were evaluated. RESULTS: Out of the 407 biopsies performed after the PCA3 test, 195 (48%) resulted positive for PCa; the PCA3 score was significantly higher in this population (p < 0.0001) differently to tPSA (p = 0.87). Moreover, the PCA3 test outperformed the f/tPSA (p = 0.01). The sensitivity (94.9) and specificity (60.1) of the PCA3 test showed a better balance for a threshold of 35 when compared to 20, even if the best result was achieved considering a cutoff of 51, with sensitivity and specificity of 82.1% and 79.3%, respectively. Finally, comparing values of the PCA3 test between two subgroups with increasing Gs (Gs ≤ 6 versus Gs ≥ 7) a significant association between PCA3 score and Gs was found (p = 0.02). CONCLUSIONS: The PCA3 test showed the best diagnostic performance when compared to tPSA and f/tPSA, facilitating the selection of high-risk patients that may benefit from the execution of a saturation prostatic biopsy. Moreover, the PCA3 test showed a prognostic value, as higher PCA3 score values are associated to a greater tumor aggressiveness.

Long-Term Response in a Patient with del(5q) Myelodysplastic Syndrome Who Discontinued Lenalidomide and Obtained a Good Response and Tolerance to Rechallenge.

BACKGROUND: The introduction of the immunomodulatory drug lenalidomide has revolutionized the treatment of patients with myelodysplastic syndromes (MDS) and deletion of the long arm of chromosome 5. Treatment with lenalidomide results in transfusion independence in the majority of patients, but some questions remain unresolved, among them the duration of treatment. Moreover, a number of unexpected long-term remissions in patients who stopped lenalidomide for various reasons have been observed. CASE REPORT: We report the case of a 60-year-old Caucasian male with deletion of the long arm of chromosome 5 and International Prognostic Scoring System (IPSS)-defined low-risk MDS who was treated with lenalidomide, achieving complete cytogenetic remission and erythroid response. After tapering off and interrupting the treatment, the patient relapsed and showed a new response by lenalidomide retreatment. Six years after the initial treatment, we registered a durable erythroid long-term response and good tolerance, but there was no evidence of a very profound cytogenetic response compared to using lenalidomide as a first-line treatment. Cytogenetic and fluorescence in situ hybridization together with hemoglobin level, mean corpuscular volume (MCV) and vitamin B12 level helped us to monitor the patient response; during the various phases of lenalidomide treatment, MCV and vitamin B12 normalization correlated with good response. CONCLUSION: Lenalidomide interruption and rechallenge in some 5q- MDS patients, with low risk according to the IPSS, is safe and feasible but does not result in a profound cytogenetic response.

Epidermal growth factor receptor gene copy number may predict lapatinib sensitivity in HER2-positive metastatic breast cancer.

OBJECTIVE: Lapatinib, a dual HER2/EGFR tyrosine kinase inhibitor (TKI), associated to capecitabine represents the treatment of choice in HER2-positive metastatic breast cancer (BC) patients in progression after trastuzumab-based therapy. Though lapatinib-based therapy prolongs the time to progression, its efficacy is often limited by the development of drug resistance. It is aimed to evaluate novel biomarkers predictive of lapatinib response, we analyzed EGFR protein and gene status in a series of 50 metastatic HER2-positive BC patients. METHODS: Lapatinib was given at 1250 mg/day continuously and capecitabine at 2000 mg/m(2)/day every 3 weeks. EGFR protein expression and gene copy number (GCN) were assessed by immunohistochemistry and FISH, respectively. Receiver operating curve (ROC) analysis identified the value of > 3.36 EGFR copies/nucleus as the cut-off point able to discriminate responders versus non-responders. RESULTS: A statistical significant correlation between EGFR GCN value > 3.36 and response to lapatinib (p = 0.01) was found. Cox regression analysis further supported these findings evidencing that HER2 score 3+ and EGFR GCN increase are positive predictor factors of lapatinib response. CONCLUSIONS: Though further investigations are needed to confirm these findings, EGFR GCN could be a suitable screening to identify the subset of BC patients particularly responsive to the dual TKI lapatinib.

EGFR molecular profiling in advanced NSCLC: a prospective phase II study in molecularly/clinically selected patients pretreated with chemotherapy.

INTRODUCTION: The optimal use of epidermal growth factor receptor (EGFR)-related molecular markers to prospectively identify tyrosine kinase inhibitor (TKI)-sensitive patients, particularly after a previous chemotherapy treatment, is currently under debate. METHODS: We designed a prospective phase II study to evaluate the activity of EGFR-TKI in four different patient groups, according to the combination of molecular (EGFR gene mutations, EGFR gene copy number and protein expression, and phosphorylated AKT expression, pAKT) and clinicopathological (histology and smoking habits) factors. Correlations between molecular alterations and clinical outcome were also explored retrospectively for first-line chemotherapy and EGFR-TKI treatment. RESULTS: Patients who had progressed during or after first-line chemotherapy were prospectively assigned to EGFR-TKI treatment as follows: (G1) EGFR mutation (n = 12); (G2) highly polysomic/amplified EGFR (n = 18); (G3) EGFR and/or pAKT positive (n = 41); (G4) adenocarcinoma/bronchoalveolar carcinoma and no smoking history (n = 15). G1 and G4 had the best and second-best overall response rate (25% and 20%, respectively), whereas the worst outcome was observed in G2 (ORR, 6%; p = 0.05). Disease control was highest in G1 and G4 (>50%) and lowest in G3 (<20%) (p = 0.02). Patients selected by EGFR mutation or clinical parameters (G1 and G4) also had significantly better progression-free survival and overall survival (p = 0.02 and p = 0.01, respectively). Multivariate analysis confirmed the impact of sex, smoking history, EGFR/KRAS mutation, and pAKT on outcomes and allowed us to derive an efficient predictive model. Histology, EGFR mutations, and pAKT were independent predictors of response to first-line chemotherapy at retrospective analysis, whereas pAKT and human epidermal growth factor receptor 2 expression were the only independent predictors of progression-free survival and overall survival. CONCLUSIONS: Selection of patients based on either EGFR mutation or clinical characteristics seems an effective approach to optimize EGFR-TKI treatment in chemotherapy-pretreated non-small-cell lung cancer patients.

Epidermal growth factor receptor gene copy number in 101 advanced colorectal cancer patients treated with chemotherapy plus cetuximab.

BACKGROUND: Responsiveness to Cetuximab alone can be mediated by an increase of Epidermal Growth factor Receptor (EGFR) Gene Copy Number (GCN). Aim of this study was to assess the role of EGFR-GCN in advanced colorectal cancer (CRC) patients receiving chemotherapy plus Cetuximab. METHODS: One hundred and one advanced CRC patients (43 untreated- and 58 pre-treated) were retrospectively studied by fluorescence in situ hybridization (FISH) to assess EGFR-GCN and by immunohistochemistry (IHC) to determine EGFR expression. Sixty-one out of 101 patients were evaluated also for k-ras status by direct sequencing. Clinical end-points were response rate (RR), progression-free survival (PFS) and overall survival (OS). RESULTS: Increased EGFR-GCN was found in 60/101 (59%) tumor samples. There was no correlation between intensity of EGFR-IHC and EGFR-GCN (p = 0.43). Patients receiving chemotherapy plus Cetuximab as first line treatment had a RR of 70% (30/43) while it was 18% (10/56) in the group with previous lines of therapy (p < 0.0001). RR was observed in 29/60 (48%) of patients with increased EGFR-GCN and in 6/28 (21%) in those without (p = 0.02). At multivariate analyses, number of chemotherapy lines and increased EGFR-GCN were predictive of response; EGFR-IHC score, increased EGFR-GCN and number of chemotherapy lines were significantly associated with a significant better PFS. Response to therapy was the only prognostic predictive factor for OS. In the 60 patients analyzed for k-ras mutations, number of chemotherapy lines, increased EGFR-GCN and k-ras wild type status predicted a better PFS. CONCLUSION: In metastatic CRC patients treated with chemotherapy plus Cetuximab number of chemotherapy lines and increased EGFR-GCN were significantly associated with a better clinical outcome, independent of k-ras status.

Genetic profile identification in clinically localized prostate carcinoma.

PURPOSE: To confirm our previously obtained results, we genetically characterized prostate cancer from patients undergo radical prostatectomy in a retrospective study. MATERIALS AND METHODS: Histological sections were evaluated for 106 patients treated with surgery from 1991 to 2004. With fluorescence in situ hybridization (FISH) method, the status of LPL (8p22), c-MYC (8q24) genes and 7, 8, X chromosomes was evaluated. RESULTS: Chromosomes 7, 8, X aneusomy was demonstrated in 91.5%, 78.3%, and 51.9% of the samples, respectively, whereas LPL deletion and MYC amplification were found in 76.0% and 1.6%. A genetic profile was considered as unfavorable when at least two aneusomic chromosomes and one altered gene were present. Tumors with an adverse genetic profile were more frequently present in patients with higher stages (P = 0.02), biochemical/clinical progression (P = 0.03), and Gleason grade 4 + 3 (P = 0.02). Multiple correspondence analysis identified one tumor group characterized by chromosome 8 aneusomy, X polysomy, LPL gene deletion, Gleason > 7 and 4 + 3 associated with progression. CONCLUSIONS: In this study, we recognized the predictive power of previously identified cytogenetic profiles. Assessment of genetic set may characterize each patient and have influence on postoperative therapeutic strategies.

Adverse genetic prognostic profiles define a poor outcome for cystectomy in bladder cancer.

Fluorescence in situ hybridization analysis was performed to evaluate P16 (9p21), P53 (17p13.1), RB1 (13q14), HER2 (17q11.2) genes and chromosomes 3, 7, 9 and 17 in 62 bladder cancer cystectomies. We found chromosome aneusomy ranged between 74.2% and 91.9%. The highest percentage of homozygous deletion was found in the P16 gene (48.4%), while the highest percentage of heterozygous deletion was in the RB1 gene (51.6%). Chromosome 17 aneusomy significantly increased in tumors with higher stage, and RB heterozygous deletion showed a higher percentage of tumors with positive lymph node. Multiple correspondence analysis (MCA) showed four different biological tumor profiles, but only one could be associated with survival, defining the most unfavorable biological profile, characterized by P53 and P16 as homozygous and heterozygous and RB as homozygous deletion. Overall survival was significantly shorter in patients with at least two positive variables out of the five detected by MCA using the Kaplan-Meier's method. The biological stratification of advanced bladder cancer patients is of particular clinical interest, because the assessment of genetic factors predictive of tumor aggressiveness may influence postoperative therapeutic strategies.

Bio-pathologic characteristics related to chromosome 11 aneusomy and cyclin D1 gene status in surgically resected stage I and II breast cancer: Identification of an adverse prognostic profile.

We aimed at developing a more detailed understanding of cyclin D1 in early stage human breast cancer and defining the biologic profiles with different prognostic value correlating cyclin D1 gene amplification and chromosome 11 aneusomy with bio-pathologic variables of known clinical importance. Cyclin D1 gene amplification and chromosome 11 aneusomy were investigated using fluorescence in situ hybridization whereas cyclin D1, PgR, HER-2, Bcl2, p53, and Ki-67 expressions were analyzed by immunohistochemistry in 121 stage I or II breast cancer patients uniformly treated with cyclophosphamide/metotrexate/5-fluorouracil-based chemotherapy. Cyclin D1 was amplified in 6.6% and overexpressed in 32.2% of cases. Amplification was not associated with any selected bio-pathologic variables, whereas the chromosome 11 aneusomy level significantly increased in tumors with higher histologic grade (P < 0.01), higher tumor size (P < 0.003), p53 nuclear accumulation (P < 0.04), and ERalpha negativity (P < 0.049). Multiple correspondence analysis showed 4 different biologic tumor profiles. The first, characterized by high Ki-67 score, p53+, cyclin D1+, HER-2+, aneusomy level > 30%, ratio (cyclin D1 gene/CEP11) > 2, was associated with tumor relapse defining the most unfavorable biologic profile. Kaplan-Meier's method showed significantly shorter disease-free survival in patients with at least 3 variables positive out of the 6 detected by multiple correspondence analysis. In multivariate analysis, the identified biologic profile emerged as the only significant prognostic indicator. Our findings are of particular clinical interest for early stage breast cancer patients, because the assessment of biologic factors predictive of tumor aggressiveness may influence postoperative therapeutic strategies.

Chromogenic in situ hybridization to detect HER-2/neu gene amplification in histological and ThinPrep-processed breast cancer fine-needle aspirates: a sensitive and practical method in the trastuzumab era.

The increasing evidence of trastuzumab efficacy in breast cancer (BC) patients means that an accurate and reproducible evaluation of HER-2 statusis of paramount importance in histological and in cytological samples. Currently, the two main methods used to analyze HER-2 amplification or overexpression are fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC). Although the two methods are strongly correlated for histological tissue, the evaluation of tumor morphology through FISH may be difficult and fluorescence fades quickly. These limitations can be overcome by chromogenic in situ hybridization (CISH), which can visualize the amplification product along with morphological features. In view of this, in the present study, we analyzed the usefulness of CISH on formalin-fixed, paraffin-embedded (FFPE) BC specimens and investigated whether CISH can be a valid technique in the determination of HER-2 status for fine-needle aspirates (FNAs) processed by liquid-based cytology. The results we obtained in a retrospective series of 111 FFPE BC specimens demonstrated good concordance between CISH and IHC and between CISH and FISH. The former concordance was comparable with that observed between FISH and IHC. When CISH was applied to a prospective series of 53 FNAs, from surgically removed BC, our data showed evidence of a higher concordance of results between liquid-based cytology and the companion FFPE tissues using CISH rather than HercepTesttrade mark. Therefore, CISH analysis, which is avaluable and reproducible alternative to FISH for selecting breast cancer patients for trastuzumab therapy, can lower false-positive immunocytochemistry findings in ThinPrep-processed FNAs.