Alternative academic approaches for testing homologous recombination deficiency in ovarian cancer in the MITO16A/MaNGO-OV2 trial.

BACKGROUND: The detection of homologous recombination deficiency (HRD) can identify patients who are more responsive to platinum and poly ADP ribose polymerase inhibitors (PARPi). MyChoice CDx (Myriad) is the most used HRD test in ovarian cancer (OC). However, some limitations of commercial tests exist, because of the high rate of inconclusive results, costs, and the impossibility of evaluating functional resistance mechanisms. PATIENTS AND METHODS: Two academic genomic tests and a functional assay, the RAD51 foci, were evaluated to detect HRD. One hundred patients with high-grade OC enrolled in the MITO16A/MaNGO-OV2 trial and treated with first-line therapy with carboplatin, paclitaxel, and bevacizumab were analyzed. RESULTS: The failure rate of the two genomic assays was 2%. The sensitivity in detecting HRD when compared with Myriad was 98.1% and 90.6%, respectively. The agreement rate with Myriad was 0.92 and 0.87, with a Cohen's κ coefficient corresponding to 0.84 and 0.74, respectively. For the RAD51 foci assay, the failure rate was 30%. When the test was successful, discordant results for deficient and proficient tumors were observed, and additional HRD patients were identified compared to Myriad; sensitivity was 82.9%, agreement rate was 0.65, and Cohen's κ coefficient was 0.18. The HRD detected by genomic assays and residual tumor at primary surgery and stage was correlated with progression-free survival at multivariate analysis. CONCLUSIONS: Results suggest the feasibility of academic tests for assessing HRD status that show robust concordance with Myriad and correlation with clinical outcome. The contribution of the functional information related to the RAD51 foci test to the genomic data needs further investigation.

A systems biology approach to investigate the mechanism of action of trabectedin in a model of myelomonocytic leukemia.

This study was designed to investigate the mode of action of trabectedin in myelomonocytic leukemia cells by applying systems biology approaches to mine gene expression profiling data and pharmacological assessment of the cellular effects. Significant enrichment was found in regulons of target genes inferred for specific transcription factors, among which MAFB was the most upregulated after treatment and was central in the transcriptional network likely to be relevant for the specific therapeutic effects of trabectedin against myelomonocytic cells. Using the Connectivity Map, similarity among transcriptional signatures elicited by treatment with different compounds was investigated, showing a high degree of similarity between transcriptional signatures of trabectedin and those of the topoisomerase I inhibitor, irinotecan, and an anti-dopaminergic antagonist, thioridazine. The study highlights the potential importance of systems biology approaches to generate new hypotheses that are experimentally testable to define the specificity of the mechanism of action of drugs.

Mechanism of action of trabectedin in desmoplastic small round cell tumor cells.

BACKGROUND: Desmoplastic small round cell tumor (DSRCT) is a rare and highly aggressive disease, that can be described as a member of the family of small round blue cell tumors. The molecular diagnostic marker is the t(11;22)(p13;q12) translocation, which creates an aberrant transcription factor, EWS-WT1, that underlies the oncogenesis of DSRCT. Current treatments are not very effective so new active drugs are needed. Trabectedin, now used as a single agent for the treatment of soft tissue sarcoma, was reported to be active in some pre-treated DSRCT patients. Using JN-DSRCT-1, a cell line derived from DSRCT expressing the EWS-WT1 fusion protein, we investigated the ability of trabectedin to modify the function of the chimeric protein, as in other sarcomas expressing fusion proteins. After detailed characterization of the EWS-WT1 transcripts structure, we investigated the mode of action of trabectedin, looking at the expression and function of the oncogenic chimera. METHODS: We characterized JN-DSRCT-1 cells using cellular approaches (FISH, Clonogenicity assay) and molecular approaches (Sanger sequencing, ChIP, GEP). RESULTS: JN-DSRCT-1 cells were sensitive to trabectedin at nanomolar concentrations. The cell line expresses different variants of EWS-WT1, some already identified in patients. EWS-WT1 mRNA expression was affected by trabectedin and chimeric protein binding on its target gene promoters was reduced. Expression profiling indicated that trabectedin affects the expression of genes involved in cell proliferation and apoptosis. CONCLUSIONS: The JN-DSRCT-1 cell line, in vitro, is sensitive to trabectedin: after drug exposure, EWS-WT1 chimera expression decreases as well as binding on its target promoters. Probably the heterogeneity of chimera transcripts is an obstacle to precisely defining the molecular mode of action of drugs, calling for further cellular models of DSRCT, possibly growing in vivo too, to mimic the biological complexity of this disease.

A prognostic regulatory pathway in stage I epithelial ovarian cancer: new hints for the poor prognosis assessment.

BACKGROUND: Clinical and pathological parameters of patients with epithelial ovarian cancer (EOC) do not thoroughly predict patients' outcome. Despite the good outcome of stage I EOC compared with that of stages III and IV, the risk assessment and treatments are almost the same. However, only 20% of stage I EOC cases relapse and die, meaning that only a proportion of patients need intensive treatment and closer follow-up. Thus, the identification of cell mechanisms that could improve outcome prediction and rationalize therapeutic options is an urgent need in the clinical practice. PATIENTS AND METHODS: We have gathered together 203 patients with stage I EOC diagnosis, from whom snap-frozen tumor biopsies were available at the time of primary surgery before any treatment. Patients, with a median follow-up of 7 years, were stratified into a training set and a validation set. RESULTS AND CONCLUSIONS: Integrated analysis of miRNA and gene expression profiles allowed to identify a prognostic cell pathway, composed of 16 miRNAs and 10 genes, wiring the cell cycle, 'Activins/Inhibins' and 'Hedgehog' signaling pathways. Once validated by an independent technique, all the elements of the circuit resulted associated with overall survival (OS) and progression-free survival (PFS), in both univariate and multivariate models. For each patient, the circuit expressions have been translated into an activation state index (integrated signature classifier, ISC), used to stratify patients into classes of risk. This prediction reaches the 89.7% of sensitivity and 96.6% of specificity for the detection of PFS events. The prognostic value was then confirmed in the external independent validation set in which the PFS events are predicted with 75% sensitivity and 94.7% specificity. Moreover, the ISC shows higher classification performance than conventional clinical classifiers. Thus, the identified circuit enhances the understanding of the molecular mechanisms lagging behind stage I EOC and the ISC improves our capabilities to assess, at the time of diagnosis, the patient risk of relapse.

Identification of high-grade serous ovarian cancer miRNA species associated with survival and drug response in patients receiving neoadjuvant chemotherapy: a retrospective longitudinal analysis using matched tumor biopsies.

BACKGROUND: Neoadjuvant chemotherapy (NACT) has been recognized as a reliable therapeutic strategy in patients with unresectable advanced epithelial ovarian cancer (EOC). The molecular events leading to platinum (Pt) response in NACT settings have hitherto not been explored. In the present work, longitudinal changes of miRNA expression profile were investigated to identify miRNA families with prognostic role in high-grade serous EOC patients who received the NACT regimen. PATIENTS AND METHODS: One hundred sixty-four matched tumor biopsies taken at initial laparoscopic evaluation and at interval-debulking surgery (IDS) after four courses of Pt-based therapy were selected from 82 stage IIIC-IV high-grade serous-EOC patients that were judged unsuitable for complete primary debulking and subjected the NACT protocol. miRNA profiling by microarray, real-time PCR and immuno-histochemical staining for Smad2 phosphorylation (P-Smad2) were used for data analysis. RESULTS: Analysis revealed that 369 miRNAs were differentially expressed in matched biopsies (referred to as DEMs). DEMs were not scattered across the genome, but clustered into families: miR-199, let-7, miR-30, miR-181 and miR-29. Multivariate analysis showed that miR-199a-3p, miR-199a-5p, miR-181a-5p and let-7g-5p associated with overall and progression-free survival (P < 0.05); miR-199a-3p, miR-199a-5p and miR-181a-5p associated with residual tumor volume and Pt-free interval (P < 0.05). Immuno-histochemical staining confirmed an enrichment of P-Smad2, a marker of transforming growth factor-ß activation, in tumors from patients with shorter PFS and OS, and with high levels of expression of miR-181a-5p (P < 0.05). Kaplan-Meier curves plotting concomitant expression of P-Smad2 and miR-181a-5p show significant differences in PFS and OS compared with those depicting the expression of each biomarker alone (P < 0.001). CONCLUSIONS: This study describes several miRNA families with a prognostic role in the NACT setting. It also confirms that concomitant analysis of P-Smad2 and miR-181a-5p in surgical samples may be capable of identifying those ovarian cancer patients with poor outcome and little chance of response to Pt-based NACT.

Profiling cancer gene mutations in longitudinal epithelial ovarian cancer biopsies by targeted next-generation sequencing: a retrospective study.

BACKGROUND: The majority of patients with stage III-IV epithelial ovarian cancer (EOC) relapse after initially responding to platinum-based chemotherapy, and develop resistance. The genomic features involved in drug resistance are unknown. To unravel some of these features, we investigated the mutational profile of genes involved in pathways related to drug sensitivity in a cohort of matched tumors obtained at first surgery (Ft-S) and second surgery (Sd-S). PATIENTS AND METHODS: Matched biopsies (33) taken at Ft-S and Sd-S were selected from the 'Pandora' tumor tissue collection. DNA libraries for 65 genes were generated using the TruSeq Custom Amplicon kit and sequenced on MiSeq (Illumina). Data were analyzed using a high-performance cluster computing platform (Cloud4CARE project) and independently validated. RESULTS: A total of 2270 somatic mutations were identified (89.85% base substitutions 8.19% indels, and 1.92% unknown). Homologous recombination (HR) genes and TP53 were mutated in the majority of Ft-S, while ATM, ATR, TOP2A and TOP2B were mutated in the entire dataset. Only 2% of mutations were conserved between matched Ft-S and Sd-S. Mutations detected at second surgery clustered patients in two groups characterized by different mutational profiles in genes associated with HR, PI3K, miRNA biogenesis and signal transduction. CONCLUSIONS: There was a low level of concordance between Ft-S and Sd-S in terms of mutations in genes involved in key processes of tumor growth and drug resistance. This result suggests the importance of future longitudinal analyses to improve the clinical management of relapsed EOC.

Mode of action of trabectedin in myxoid liposarcomas.

To elucidate the mechanisms behind the high sensitivity of myxoid/round cell liposarcoma (MRCL) to trabectedin and the suggested selectivity for specific subtypes, we have developed and characterized three MRCL xenografts, namely ML017, ML015 and ML004 differing for the break point of the fusion gene FUS-CHOP, respectively of type I, II and III. FUS-CHOP binding to the promoters of some target genes such as Pentraxin 3 or Fibronectin 1, assessed by chromatin immunoprecipitation, was strongly reduced in the tumor 24 h after the first or the third weekly dose of trabectedin, indicating that the drug at therapeutic doses causes a detachment of the FUS-CHOP chimera from its target promoters as previously shown in vitro. Moreover, the higher sensitivity of MRCL types I and II appears to be related to a more prolonged block of the transactivating activity of the fusion protein. Doxorubicin did not affect the binding of FUS-CHOP to target promoters. Histologically, the response to trabectedin in ML017 and ML015 was associated with a marked depletion of non-lipogenic tumoral cells and vascular component, as well as lipidic maturation as confirmed by PPARγ2 expression in western Blot. By contrast, in ML004 no major changes either in the cellularity or in the amount of mature were found, and consistently PPARγ2 was null. In conclusion, the data support the view that the selective mechanism of action of trabectedin in MRCL is specific and related to its ability to cause a functional inactivation of the oncogenic chimera with consequent derepression of the adypocytic differentiation.

Pharmacological blockade of IL-1ß/IL-1 receptor type 1 axis during epileptogenesis provides neuroprotection in two rat models of temporal lobe epilepsy.

We studied whether pharmacological blockade of the IL-1ß-mediated signaling, rapidly activated in forebrain by epileptogenic injuries, affords neuroprotection in two different rat models of status epilepticus (SE). As secondary outcome, we measured treatment's effect on SE-induced epileptogenesis. IL-1ß signaling was blocked by systemic administration of two antiinflammatory drugs, namely human recombinant IL-1 receptor antagonist (anakinra), the naturally occurring and clinically used competitive IL-1 receptor type 1 antagonist, and VX-765 a specific non-peptide inhibitor of IL-1ß cleavage and release. Antiinflammatory drugs were given 60min after antiepileptic (AED) drug-controlled SE induced by pilocarpine, or 180min after unrestrained electrical SE, for 7days using a protocol yielding therapeutic drug levels in brain. This drug combination significantly decreased both IL-1ß expression in astrocytes and cell loss in rat forebrain. Neuroprotection and the antiinflammatory effect were more pronounced in the electrical SE model. Onset of epilepsy, and frequency and duration of seizures 3months after electrical SE were not significantly modified. Transcriptomic analysis in the hippocampus showed that the combined treatment did not affect the broad inflammatory response induced by SE during epileptogenesis. In particular, the treatment did not prevent the induction of the complement system and Toll-like receptors, both contributing to cell loss and seizure generation. We conclude that the IL-1ß signaling represents an important target for reducing cell loss after SE. The data highlight a new class of clinically tested agents affording neuroprotection after a delayed post-injury intervention. Earlier blockade of this rapid onset inflammatory pathway during SE, or concomitant treatment with antiinflammatory drugs targeting additional components of the broad inflammatory response to SE, or co-treatment with AEDs, is likely to be required for optimizing beneficial outcomes.

Influence of KIR genes and their HLA ligands in susceptibility to dengue in a population from southern Brazil.

Killer cell immunoglobulin-like receptors (KIR) form a group of regulatory molecules that specifically recognise human leukocyte antigen (HLA) class I molecules, modulating the cytolytic activity of natural killer cells. The purpose of this study was to investigate the influence of KIR genes and their class I HLA ligands in susceptibility to dengue fever in a population from southern Brazil through a case-control study. One hundred four subjects with confirmed diagnoses of dengue participated in this study, along with a control group of 172 individuals from the same geographic area. HLA and KIR genotyping was performed by polymerase chain reaction with sequence-specific oligonucleotide probes (PCR-SSOP) and with sequence-specific primer (PCR-SSP) techniques, respectively. Data analysis showed significant differences for the KIR2DS1 (54.8% vs 40.7%, P = 0.03), KIR2DS5 (50.0% vs 36.0%, P = 0.03) and KIR2DL5 (76.0% vs 56.4%, P = 0.001) genes. With regard to KIR-ligand pairs, positive associations with dengue were observed in KIR3DS1-Bw4 (45.2% vs 29.7%, P = 0.01), KIR3DL1-Bw4 (80.7% vs 65.1%, P < 0.001), KIR2DL1-C2 (75.0% vs 62.2%, P = 0.03) and KIR2DS1-C2 (40.4% vs 25.6%, P = 0.01) interactions, and a negative association in KIR2DL3-C1/C1 (18.2% vs 33.1%, P = 0.01). Furthermore, the analysis of KIR haplogroups showed a possible protective factor against dengue fever in individuals with the AA genotype. Taken together, these results suggest the existence of genetic predisposition to dengue fever in the population from southern Brazil.

Algorithm for automatic genotype calling of single nucleotide polymorphisms using the full course of TaqMan real-time data.

Single nucleotide polymorphisms (SNPs) are often determined using TaqMan real-time PCR assays (Applied Biosystems) and commercial software that assigns genotypes based on reporter probe signals at the end of amplification. Limitations to the large-scale application of this approach include the need for positive controls or operator intervention to set signal thresholds when one allele is rare. In the interest of optimizing real-time PCR genotyping, we developed an algorithm for automatic genotype calling based on the full course of real-time PCR data. Best cycle genotyping algorithm (BCGA), written in the open source language R, is based on the assumptions that classification depends on the time (cycle) of amplification and that it is possible to identify a best discriminating cycle for each SNP assay. The algorithm is unique in that it classifies samples according to the behavior of blanks (no DNA samples), which cluster with heterozygous samples. This method of classification eliminates the need for positive controls and permits accurate genotyping even in the absence of a genotype class, for example when one allele is rare. Here, we describe the algorithm and test its validity, compared to the standard end-point method and to DNA sequencing.