Treatment Stratification Of Patients Research Articles

Chromatin Conformation Signatures Associated with Epigenetic Deregulation of the FIP1L1 and PDGFRA Genes

The human genome is organized into topological domains with discrete structural and regulatory units that influence gene expression epigenetically (Crutchley et al., 2010). In glioma patients with IDH1 (R132H) mutations, researchers detected aberrant methylation of transcriptional CTCF-enhancer regions between the FIPL1L1 and PDGFRA genes. Conformational changes to the chromosomal arrangement of these regions have been shown to upregulate PDGFRA expression (Flavahan et al; 2016). Flavahan et al also demonstrated the down regulation of cell growth and proliferation in the glioma cells with the addition of tyrosine kinase inhibitors that target the interaction.We explored this epigenetic interaction and its utility in the stratification of patients for treatment with tyrosine kinase inhibitors. Using a high-resolution chromosome-conformation capture or 3C analysis platform known as EpiSwitchTMand quantitative PCR, we mapped, evaluated, and quantified the conformational juxtaposition between FIP1L1 and PDGFRA in glioma-cell lines with and without IDH mutations. Deregulation of PDGFRA by interstitial deletion at 4q12 and fusion to FIP1L1 associated with chronic esosinophic leukemias prompted our group to also investigate whether the same chromosome-conformation interactions are present in EOL-1 and other leukemic cell lines.EpiSwitchTM templates were produced using a modified process to purify the 3C DNA. Unlike previous protocols, only one set of primers is required. Chromosome conformation capture analysis with a probe, single step PCR and gel purification was used to identify and sequence 3C interactions in the AML cell lines EOL-1 and HL-60. qPCR templates, adjusted to 20 ng of 3C library DNA, were used with concentration-matched negative controls (ie, 3C libraries derived from adipose biopies and normal blood). A 3C interaction with MMP-1 was used as a internal control for the EpiSwitchTM library. A dual label hydrolysis probe was used to detect the sequenced interaction. Five novel interactions were identified between FIP1L1 and PDGFRA by single step PCR and confirmed by sequencing. We quantified one interaction with a dual-labeled hydrolysis probe based qPCR assay to determine copy number in eight cell lines: BT-412 (anaplastic oligoastrocytoma), DBTRG-05MG (glioblastoma multiforme), U-3T3 (glioblastoma astrocytoma), U-87 (glioblastoma astrocytoma), GDM-1 (acute myelomonocytic leukemia), EOL-1 (acute myeloid leukemia, AML), HL-60 (AML) and KG-1 (AML).Using EpiSwitchTM and ISO standard MIQE-compliant qPCR design, five specific epigenetic interactions were detected in three types of cancerous cell lines including EOL-1 and HL-60. qPCR was used to detect one specific difference between normal blood (n = 4) and leukemic cell lines (n = 4). By qPCR the interaction was detected in two AML cell lines known to be highly sensitive to the tyrosine kinase inhibitor Glivec (EOL-1 and GDM-1), as well as HL-60 cells, but not in KG-1. The FIP1L1 PDGFRA 3C interaction was also found in DBTRG-05MG and U-373 at a similar copy number compared to the AML cell lines. A glioma patient biopsy tissue also tested positive for the conformational-chromosome interaction by qPCR compared with an adipose biopsy control. An interaction in the MMP-1 gene was used as an internal positive control in all samples tested.Emerging epigenetic approaches, such as chromosome conformation signatures as detected by EpiSwitchTM, are providing sensitive and accurate methods for improving patient treatment stratification. Detection of interactions between PDGFRA and FIP1L1 based on chromosomal conformational changes have the potential to improve upon the available methods for identifying patients who may benefit from treatment with PDGFRA-targeted tyrosine kinase inhibitors. Further research is needed to validate the method described using blood and biopsy samples in patients with glioma, AML and other myeloproliferative neoplasms associated with the FIP1L1-PDGFRA fusion gene. DisclosuresGrand:Oxford BioDynamics Limited: Employment, Equity Ownership. Bird:Oxford BioDynamics Limited: Employment. Corfield:Oxford BioDynamics Limited: Employment. Dezfouli:Oxford BioDynamics Limited: Employment. Warren:Oxford BioDynamics Limited: Employment. Foulkes:Oxford BioDynamics Limited: Employment. Khudari:Oxford BioDynamics Limited: Employment. Salter:Oxford BioDynamics Limited: Employment. Mahecha:Oxford BioDynamics Limited: Employment. Ssentongo:Oxford BioDynamics Limited: Employment. Green:Oxford BioDynamics Limited: Employment, Equity Ownership. Womersley:Oxford BioDynamics Limited: Employment, Patents & Royalties. Hunter:Oxford BioDynamics Limited: Employment. Ramadass:Oxford BioDynamics Limited: Employment. Akoulitchev:Oxford BioDynamics Limited: Employment, Equity Ownership.

Biopsy proportion of tumour predicts pathological tumour response and benefit from chemotherapy in resectable oesophageal carcinoma: results from the UK MRC OE02 trial.

BackgroundNeoadjuvant chemotherapy followed by surgery is the standard of care for UK patients with locally advanced resectable oesophageal carcinoma (OeC). However, not all patients benefit from multimodal treatment and there is a clinical need for biomarkers which can identify chemotherapy responders. This study investigated whether the proportion of tumour cells per tumour area (PoT) measured in the pre-treatment biopsy predicts chemotherapy benefit for OeC patients.Patients and methodsPoT was quantified using digitized haematoxylin/eosin stained pre-treatment biopsy slides from 281 OeC patients from the UK MRC OE02 trial (141 treated by surgery alone (S); 140 treated by 5-fluorouracil/cisplatin followed by surgery (CS)). The relationship between PoT and clinicopathological data including tumour regression grade (TRG), overall survival and treatment interaction was investigated.ResultsPoT was associated with chemotherapy benefit in a non-linear fashion (test for interaction, P=0.006). Only patients with a biopsy PoT between 40% and 70% received a significant survival benefit from neoadjuvant chemotherapy (N=129; HR (95%CI):1.94 (1.39-2.71), unlike those with lower or higher PoT (PoT<40%, N=39, HR:1.25 (0.66-2.35); PoT>70% (N=28, HR:0.65 (0.36-1.18)). High pre-treatment PoT was related to lack of primary tumour regression (TRG 4 or 5), P=0.0402.ConclusionsThis is the first study to identify in a representative subgroup of OeC patients from a large randomized phase III trial that the proportion of tumour in the pre-chemotherapy biopsy predicts benefit from chemotherapy and may be a clinically useful biomarker for patient treatment stratification.

DrugTargetInspector: An assistance tool for patient treatment stratification

DrugTargetInspector: An assistance tool for patient treatment stratification

Radiomics Analysis on FLT-PET/MRI for Characterization of Early Treatment Response in Renal Cell Carcinoma: A Proof-of-Concept Study

Studying early response to cancer treatment is significant for patient treatment stratification and follow-up. Although recent advances in positron emission tomography (PET) and magnetic resonance imaging (MRI) allow for evaluation of tumor response, a quantitative objective assessment of treatment-related effects offers localization and quantification of structural and functional changes in the tumor region. Radiomics, the process of computerized extraction of features from radiographic images, is a new strategy for capturing subtle changes in the tumor region that works by quantifying subvisual patterns which might escape human identification. The goal of this study was to demonstrate feasibility for performing radiomics analysis on integrated PET/MRI to characterize early treatment response in metastatic renal cell carcinoma (RCC) undergoing sunitinib therapy. Two patients with advanced RCC were imaged using an integrated PET/MRI scanner. [18 F] fluorothymidine (FLT) was used as the PET radiotracer, which can measure the degree of cell proliferation. Image acquisitions included test/retest scans before sunitinib treatment and one scan 3 weeks into treatment using [18 F] FLT-PET, T2-weighted (T2w), and diffusion-weighted imaging (DWI) protocols, where DWI yielded an apparent diffusion coefficient (ADC) map. Our framework to quantitatively characterize treatment-related changes involved the following analytic steps: 1) intraacquisition and interacquisition registration of protocols to allow voxel-wise comparison of changes in radiomic features, 2) correction and pseudoquantification of T2w images to remove acquisition artifacts and examine tissue-specific response, 3) characterization of information captured by T2w MRI, FLT-PET, and ADC via radiomics, and 4) combining multiparametric information to create a map of integrated changes from PET/MRI radiomic features. Standardized uptake value (from FLT-PET) and ADC textures ranked highest for reproducibility in a test/retest evaluation as well as for capturing treatment response, in comparison to high variability seen in T2w MRI. The highest-ranked radiomic feature yielded a normalized percentage change of 63% within the RCC region and 17% in a spatially distinct normal region relative to its pretreatment value. By comparison, both the original and postprocessed T2w signal intensity appeared to be markedly less sensitive and specific to changes within the tumor. Our preliminary results thus suggest that radiomics analysis could be a powerful tool for characterizing treatment response in integrated PET/MRI.

Magnetic resonance analysis of malignant transformation in recurrent glioma

BackgroundPatients with low-grade glioma (LGG) have a relatively long survival, and a balance is often struck between treating the tumor and impacting quality of life. While lesions may remain stable for many years, they may also undergo malignant transformation (MT) at the time of recurrence and require more aggressive intervention. Here we report on a state-of-the-art multiparametric MRI study of patients with recurrent LGG.MethodsOne hundred and eleven patients previously diagnosed with LGG were scanned at either 1.5 T or 3 T MR at the time of recurrence. Volumetric and intensity parameters were estimated from anatomic, diffusion, perfusion, and metabolic MR data. Direct comparisons of histopathological markers from image-guided tissue samples with metrics derived from the corresponding locations on the in vivo images were made. A bioinformatics approach was applied to visualize and interpret these results, which included imaging heatmaps and network analysis. Multivariate linear-regression modeling was utilized for predicting transformation.ResultsMany advanced imaging parameters were found to be significantly different for patients with tumors that had undergone MT versus those that had not. Imaging metrics calculated at the tissue sample locations highlighted the distinct biological significance of the imaging and the heterogeneity present in recurrent LGG, while multivariate modeling yielded a 76.04% accuracy in predicting MT.ConclusionsThe acquisition and quantitative analysis of such multiparametric MR data may ultimately allow for improved clinical assessment and treatment stratification for patients with recurrent LGG.

Segmental distribution of some common molecular markers for colorectal cancer (CRC): influencing factors and potential implications.

Proximal and distal colorectal cancers (CRCs) are regarded as distinct disease entities, evolving through different genetic pathways and showing multiple clinicopathological and molecular differences. Segmental distribution of some common markers (e.g., KRAS, EGFR, Ki-67, Bcl-2, COX-2) is clinically important, potentially affecting their prognostic or predictive value. However, this distribution is influenced by a variety of factors such as the anatomical overlap of tumorigenic molecular events, associations of some markers with other clinicopathological features (stage and/or grade), and wide methodological variability in markers' assessment. All these factors represent principal influences followed by intratumoral heterogeneity and geographic variation in the frequency of detection of particular markers, whereas the role of other potential influences (e.g., pre-adjuvant treatment, interaction between markers) remains rather unclear. Better understanding and elucidation of the various influences may provide a more accurate picture of the segmental distribution of molecular markers in CRC, potentially allowing the application of a novel patient stratification for treatment, based on particular molecular profiles in combination with tumor location.

DrugTargetInspector: An assistance tool for patient treatment stratification.

Cancer is a large class of diseases that are characterized by a common set of features, known as the Hallmarks of cancer. One of these hallmarks is the acquisition of genome instability and mutations. This, combined with high proliferation rates and failure of repair mechanisms, leads to clonal evolution as well as a high genotypic and phenotypic diversity within the tumor. As a consequence, treatment and therapy of malignant tumors is still a grand challenge. Moreover, under selective pressure, e.g., caused by chemotherapy, resistant subpopulations can emerge that then may lead to relapse. In order to minimize the risk of developing multidrug-resistant tumor cell populations, optimal (combination) therapies have to be determined on the basis of an in-depth characterization of the tumor's genetic and phenotypic makeup, a process that is an important aspect of stratified medicine and precision medicine. We present DrugTargetInspector (DTI), an interactive assistance tool for treatment stratification. DTI analyzes genomic, transcriptomic, and proteomic datasets and provides information on deregulated drug targets, enriched biological pathways, and deregulated subnetworks, as well as mutations and their potential effects on putative drug targets and genes of interest. To demonstrate DTI's broad scope of applicability, we present case studies on several cancer types and different types of input -omics data. DTI's integrative approach allows users to characterize the tumor under investigation based on various -omics datasets and to elucidate putative treatment options based on clinical decision guidelines, but also proposing additional points of intervention that might be neglected otherwise. DTI can be freely accessed at http://dti.bioinf.uni-sb.de.

The influence of composition and location on the toughness of human atherosclerotic femoral plaque tissue

The toughness of femoral atherosclerotic tissue is of pivotal importance to understanding the mechanism of luminal expansion during cutting balloon angioplasty (CBA) in the peripheral vessels. Furthermore, the ability to relate this parameter to plaque composition, pathological inclusions and location within the femoral vessels would allow for the improvement of existing CBA technology and for the stratification of patient treatment based on the predicted fracture response of the plaque tissue to CBA. Such information may lead to a reduction in clinically observed complications, an improvement in trial results and an increased adoption of the CBA technique to reduce vessel trauma and further endovascular treatment uptake.This study characterises the toughness of atherosclerotic plaque extracted from the femoral arteries of ten patients using a lubricated guillotine cutting test to determine the critical energy release rate. This information is related to the location that the plaque section was removed from within the femoral vessels and the composition of the plaque tissue, determined using Fourier Transform InfraRed spectroscopy, to establish the influence of location and composition on the toughness of the plaque tissue. Scanning electron microscopy (SEM) is employed to examine the fracture surfaces of the sections to determine the contribution of tissue morphology to toughness.Toughness results exhibit large inter and intra patient and location variance with values ranging far above and below the toughness of healthy porcine arterial tissue (Range: 1330–3035 for location and 140–4560J/m2 for patients). No significant difference in mean toughness is observed between patients or location. However, the composition parameter representing the calcified tissue content of the plaque correlates significantly with sample toughness (r=0.949, p<0.001). SEM reveals the presence of large calcified regions in the toughest sections that are absent from the least tough sections. Regression analysis highlights the potential of employing the calcified tissue content of the plaque as a preoperative tool for predicting the fracture response of a target lesion to CBA (R2=0.885, p<0.001). Statement of SignificanceThis study addresses a gap in current knowledge regarding the influence of plaque location, composition and morphology on the toughness of human femoral plaque tissue. Such information is of great importance to the continued improvement of endovascular treatments, particularly cutting balloon angioplasty (CBA), which require experimentally derived data as a framework for assessing clinical cases and advancing medical devices. This study identifies that femoral plaque tissue exhibits large inter and intra patient and location variance regarding tissue toughness. Increasing calcified plaque content is demonstrated to correlate significantly with increasing toughness. This highlights the potential for predicting target lesion toughness which may lead to an increased adoption of the CBA technique and also further the uptake of endovascular treatment.

Plectin as a prognostic marker in non-metastatic oral squamous cell carcinoma.

BackgroundOral squamous cell carcinoma (OSCC) is associated with a poor 5-year survival rate. In general, patients diagnosed with small tumors have a fairly good prognosis, but some small tumors have an aggressive behavior leading to early death. There are at present no reliable prognostic biomarkers for oral cancers. Thus, to optimize treatment for the individual patient, there is a need for biomarkers that can predict tumor behavior.MethodIn the present study the potential prognostic value of plectin was evaluated by a tissue microarray (TMA) based immunohistochemical analysis of primary tumor tissue obtained from a North Norwegian cohort of 115 patients diagnosed with OSCC. The expression of plectin was compared with clinicopathological variables and 5 year survival.ResultsThe statistical analysis revealed that low expression of plectin in the tumor cells predicted a favorable outcome for patients with non-metastatic disease (p = 0.008). Furthermore, the expression of plectin was found to correlate (p = 0.01) with the expression of uPAR, which we have previously found to be a potential prognostic marker for T1N0 tumors.ConclusionsOur results indicate that low expression of plectin predicts a favorable outcome for patients with non-metastatic OSCC and the expression level of plectin may therefore be used in the treatment stratification for patients with early stage disease.

MicroRNA-330-5p as a Putative Modulator of Neoadjuvant Chemoradiotherapy Sensitivity in Oesophageal Adenocarcinoma.

Oesophageal adenocarcinoma (OAC) is the sixth most common cause of cancer deaths worldwide, and the 5-year survival rate for patients diagnosed with the disease is approximately 17%. The standard of care for locally advanced disease is neoadjuvant chemotherapy or, more commonly, combined neoadjuvant chemoradiation therapy (neo-CRT) prior to surgery. Unfortunately, ~60-70% of patients will fail to respond to neo-CRT. Therefore, the identification of biomarkers indicative of patient response to treatment has significant clinical implications in the stratification of patient treatment. Furthermore, understanding the molecular mechanisms underpinning tumour response and resistance to neo-CRT will contribute towards the identification of novel therapeutic targets for enhancing OAC sensitivity to CRT. MicroRNAs (miRNA/miR) function to regulate gene and protein expression and play a causal role in cancer development and progression. MiRNAs have also been identified as modulators of key cellular pathways associated with resistance to CRT. Here, to identify miRNAs associated with resistance to CRT, pre-treatment diagnostic biopsy specimens from patients with OAC were analysed using miRNA-profiling arrays. In pre-treatment biopsies miR-330-5p was the most downregulated miRNA in patients who subsequently failed to respond to neo-CRT. The role of miR-330 as a potential modulator of tumour response and sensitivity to CRT in OAC was further investigated in vitro. Through vector-based overexpression the E2F1/p-AKT survival pathway, as previously described, was confirmed as a target of miR-330 regulation. However, miR-330-mediated alterations to the E2F1/p-AKT pathway were insufficient to significantly alter cellular sensitivity to chemotherapy (cisplatin and 5-flurouracil). In contrast, silencing of miR-330-5p enhanced, albeit subtly, cellular resistance to clinically relevant doses of radiation. This study highlights the need for further investigation into the potential of miR-330-5p as a predictive biomarker of patient sensitivity to neo-CRT and as a novel therapeutic target for manipulating cellular sensitivity to neo-CRT in patients with OAC.

The need for a personalized approach for prostate cancer management.

The stratification of patients for treatment of prostate cancer is based on very general parameters like prostate-specific antigen, Gleason score, and TNM classification. We use these rough parameters for selection of active surveillance, active treatment, and even for the treatment selection in metastasized or castration-resistant prostate cancers. Up to now, we have not used individualized genetic classifiers for detailed sub-stratification, thus treating all patients as equal, and being only moderately successful. With the expected increase in systemic treatments, there is an apparent need for such classifiers. We will address these needs in this short commentary.

Correlation between the International Neuroblastoma Pathology Classification and genomic signature in neuroblastoma.

The International Neuroblastoma Pathology Classification (INPC) has a prognostic impact that distinguishes two categories of neuroblastoma: favorable histology (FH) and unfavorable histology (UH). We analyzed 92 cases of neuroblastoma with the INPC evaluation and genomic grouping to investigate the correlation between the INPC and genomic signature, together with their prognostic significance. The correlation of UH tumor and partial gains and/or losses (GGP), as well as the correlation of FH tumor and whole gains and/or losses (GGW), was statistically significant. Both UH and GGP were late-onset (median age at diagnosis was 36 and 48 months, respectively) and had poor prognosis (overall survival rate [OS], 43.1% and 42.4%, respectively). In contrast, both FH and GGW were early-onset (median age at diagnosis, 4 and 9.5 months, respectively) and had favorable prognosis (OS, 88.6% and 87.1%, respectively). Unfavorable histology and GGP had significantly inferior OS compared to FH and GGW. Overall survival was not significantly different among the genomic groups in FH; however, it was inferior in UH with GGP. In UH with a single copy MYCN, genomic subgroups GGP2s (both 1p and 11q losses) and GGP3s (partial 11q loss but not 1p loss) indicated significantly poor prognosis compared to GGP4s (no partial 1p and 11q loss). As INPC and MYCN amplification were found to be the most powerful prognostic biological factors, they should be included with genomic grouping as treatment stratification for patients with UH and single copy of MYCN.

Low RBM3 protein expression correlates with clinical stage, prognostic classification and increased risk of treatment failure in testicular non-seminomatous germ cell cancer.

BackgroundExpression of the RNA-binding motif protein 3 (RBM3) has been shown to correlate with favourable clinicopathological parameters and prognosis in several cancer diseases. The aim of this study was to examine the expression and prognostic ability of RBM3 in patients with testicular non-seminomatous germ cell tumours (NSGCT).Patients and MethodsImmunohistochemical RBM3 expression was analysed in tissue microarrays with tumours from 206 patients. Chi-square test was applied to analyze associations between RBM3 expression and clinicopathological parameters. Kaplan-Meier analysis was used to assess the impact of RBM3 expression on cancer-specific survival (CSS) and failure-free survival (FFS). Cox regression proportional hazards models were used to estimate the relative risk for failure.ResultsIn the entire cohort, there was a significant association between clinical stage (p=0.044) and RBM3 expression. Weak RBM3 expression correlated with a significantly reduced FFS [79.3% versus 90.4% (p=0.019)] and CSS [87.5% versus 97.3% (p=0.047)]. For patients with metastatic disease (n = 88), significant associations were found between RBM3 expression and IGCCC group (p=0.007). The FFS was significantly inferior for patients with low tumour-specific RBM3 expression [59.3% versus 79.0% (p=0.013)], and this association remained significant in a multivariable model for patients with metastatic disease (HR=3.67; 95% CI 1.14, 11.89).ConclusionLow RBM3 expression is an independent predictor of treatment failure in metastatic NSGCT, in relation to the prognostic factors included in the International Germ Cell Consensus Classification (IGCCC). These findings suggest that RBM3 may be a potential biomarker for treatment stratification in patients with metastatic non-seminomatous germ cell tumours, and therefore merit further validation.

Prognostic and treatment predictive significance of SATB1 and SATB2 expression in pancreatic and periampullary adenocarcinoma.

BackgroundPancreatic cancer and other pancreaticobiliary type periampullary adenocarcinomas have a dismal prognosis even after resection and neoadjuvant chemotherapy. Intestinal type periampullary adenocarcinomas generally have a better prognosis, but little is known on optimal neoadjuvant and adjuvant treatment. New prognostic and treatment predictive biomarkers are needed for improved treatment stratification of patients with both types of periampullary adenocarcinoma. Expression of the Special AT-rich sequence-binding protein 1 (SATB1) has been demonstrated to confer a worse prognosis in several tumour types, whereas its close homologue SATB2 is a proposed diagnostic and favourable prognostic marker for colorectal cancer. The prognostic value of SATB1 and SATB2 expression in periampullary adenocarcinoma has not yet been described.MethodsImmunohistochemical expression of SATB1 and SATB2 was analysed in tissue microarrays with primary tumours and a subset of paired lymph node metastases from 175 patients operated with pancreaticoduodenectomy for periampullary adenocarcinoma. Kaplan-Meier and Cox regression analysis were applied to explore the impact of SATB1 and SATB2 expression on recurrence free survival (RFS) and overall survival (OS).ResultsPositive expression of SATB1 was denoted in 16/106 primary pancreatobiliary type tumours and 11/65 metastases, and in 15/63 primary intestinal type tumours and 4/26 metastases, respectively. Expression of SATB1 was an independent predictor of a significantly shorter RFS and OS in pancreatobiliary type, but not in intestinal type adenocarcinomas. Moreover, SATB1 expression predicted an improved response to adjuvant chemotherapy in both tumour types. SATB2-expression was seen in 3/107 pancreatobiliary type primary tumours, and in 8/61 intestinal type primary tumours. The small number of cases with positive SATB2 expression did not allow for any firm conclusions on its prognostic value.ConclusionsThese findings demonstrate the potential utility of SATB1 as a prognostic and predictive biomarker for chemotherapy response in both intestinal type and pancreatobiliary type periampullary adenocarcinomas, including pancreatic cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-014-0289-8) contains supplementary material, which is available to authorized users.

Low Rbm3 Protein Expression Correlates with Clinical Stage, Prognostic Index and Increased Risk of Treatment Failure in Testicular Non-Seminomatous Germ Cell Cancer

ABSTRACT Aim: Expression of the RNA-binding motif protein 3 (RBM3) has in previous studies been shown to correlate with favourable clinicopathological parameters and prognosis in a number of cancer forms. The aim of this study was to examine the expression and prognostic ability of RBM3 in patients with non-seminomatous germ cell tumours (NSGCT). Methods: Immunohistochemical RBM3 expression was analysed in tissue microarrays with tumours from 206 patients with NSGCT. Chi-square test was applied to analyze associations between RBM3 expression and clinicopathological parameters. Kaplan-Meier analysis was used to assess the impact of RBM3 expression on cancer-specific survival (CSS) and progression-free survival (PFS). Cox regression proportional hazards models were used to estimate the relative risk for progression in both uni- and multivariable analysis. Results: In the entire cohort, there was a significant association between clinical stage (p=0.044) and RBM3 expression. Low RBM3 expression correlated with a significantly reduced PFS [67.7% versus 87.8% (p=0.001)] and CSS [87.5% versus 97.3% (p=0.047)]. For patients with metastatic disease (n=88), significant associations were found between RBM3 expression and IGCCC prognostic index (p=0.007), combined tumor marker status (p=0.001) and HCG level (p=0.010). The PFS was significantly inferior for patients with low tumour-specific RBM3 expression [40.0% versus 73.0% (p=0.002)], and this association remained significant in a multivariable model (HR=2.98; 95% CI 1.14, 7.75). Conclusions: Low RBM3 expression is significantly associated with a worse IGCCC-prognostic index and is an independent predictor of disease progression in metastatic NSGCT. These findings suggest that RBM3 may be a potential biomarker for treatment stratification in patients with metastatic non-seminomatous germ cell tumours, and therefore merit further validation. Disclosure: All authors have declared no conflicts of interest.

COX-2 expression is predictive for early relapse and aromatase inhibitor resistance in patients with ductal carcinoma in situ of the breast, and is a target for treatment.

Background:Stratification of patients for treatment of ductal carcinoma in situ (DCIS) is suboptimal, with high systemic overtreatment rates.Methods:A training set of 95 tumours from women with pure DCIS were immunostained for proteins involved in cell survival, hypoxia, growth factor and hormone signalling. A generalised linear regression with regularisation and variable selection was applied to a multiple covariate Cox survival analysis with recurrence-free survival 10-fold cross-validation and leave-one-out iterative approach were used to build and test the model that was validated using an independent cohort of 58 patients with pure DCIS. The clinical role of a COX-2-targeting agent was then tested in a proof-of-concept neoadjuvant randomised trial in ER-positive DCIS treated with exemestane 25 mg day−1±celecoxib 800 mg day−1.Results:The COX-2 expression was an independent prognostic factor for early relapse in the training (HR 37.47 (95% CI: 5.56–252.74) P=0.0001) and independent validation cohort (HR 3.9 (95% CI: 1.8–8.3) P=0.002). There was no significant interaction with other clinicopathological variables. A statistically significant reduction of Ki-67 expression after treatment with exemestane±celecoxib was observed (P<0.02) with greater reduction in the combination arm (P<0.004). Concomitant reduction in COX-2 expression was statistically significant in the exemestane and celecoxib arm (P<0.03) only.Conclusions:In patients with DCIS, COX-2 may predict recurrence, aiding clinical decision making. A combination of an aromatase inhibitor and celecoxib has significant biological effect and may be integrated into treatment of COX2-positive DCIS at high risk of recurrence.

Fibroblastic growth factor receptor 1 amplification in osteosarcoma is associated with poor response to neo-adjuvant chemotherapy.

Osteosarcoma, the most common primary bone sarcoma, is a genetically complex disease with no widely accepted biomarker to allow stratification of patients for treatment. After a recent report of one osteosarcoma cell line and one tumor exhibiting fibroblastic growth factor receptor 1 (FGFR1) gene amplification, the aim of this work was to assess the frequency of FGFR1 amplification in a larger cohort of osteosarcoma and to determine if this biomarker could be used for stratification of patients for treatment. About 352 osteosarcoma samples from 288 patients were analyzed for FGFR1 amplification by interphase fluorescence in situ hybridization. FGFR1 amplification was detected in 18.5% of patients whose tumors revealed a poor response to chemotherapy, and no patients whose tumors responded well to therapy harbored this genetic alteration. FGFR1 amplification is present disproportionately in the rarer histological variants of osteosarcoma. This study provides a rationale for inclusion of patients with osteosarcoma in clinical trials using FGFR kinase inhibitors.

Cancer molecular pathobiology in the clinics: concluding remarks.

This interesting issue in which the Institute of Oncology “Prof. Dr. Ion Chiricuta” of Cluj and the Cancer Institute “G. Paolo II” of Bari approach the problem of cancer biomarkers from different perspectives represents an attempt to provide a novel view of biological, clinical and methodological innovative aspects in this field. In fact, there is a critical need for reliable biomarkers in cancer in order to improve (i) early detection, (ii) accuracy of prognosis, (iii) patient stratification for treatments, and (iv) monitoring of tumor response and relapse. Although technological advances in the field of genomics, metabolomics, and proteomics have resulted in the identification of many biomarker candidates, only a few of them have been approved and display a value in clinical practice. This is particularly true for biomarkers suitable for early diagnosis of cancers, despite the considerable efforts devoted to this goal. In the field of diagnosis, one important challenge is getting biomarkers specific enough to avoid false positive conclusions that lead to unnecessarily expensive (imaging) and invasive (biopsies) interventions. Although novel technologies have produced detailed information on the molecular aspects of cancer biology, for the most part, the specificity displayed by single or multiple biomarkers is insufficient to allow the assays to be used in large populations. Very sensitive techniques to address specific hallmarks of cancer and combined approaches (metabolomics and imaging, for instance) need to be improved in the future in order to succeed in this objective. Next-generation sequencing techniques and especially RNAseq provide data about cancer-specific splice variants that can be developed as very useful biomarkers, either by detection of the variants as circulating DNA or by using antibodies when the splice variant results in novel epitopes in transmembrane or soluble proteins. In terms of prognosis and prediction of response, the situation is more favorable because of access to tumor biopsies, which allows the evaluation of different biomarkers at the protein, RNA or DNA levels. A number of signatures are commercialized or under evaluation in large clinical trials and will help clinicians to make therapeutic decisions. In the case of gene profiling in breast cancer, patients who can benefit from adjuvant chemotherapy can be selected based on a high risk of local relapse/ metastasis or to exclude low-risk patients from unnecessary therapeutic intervention. In the era of evidence-based medicine, there is an emergence of biomarkers predicting the response to targeted therapy. These biomarkers allow the selection of the patients who would or would not benefit from a given treatment. Determination of HER2 expression or KRAS mutations are mandatory for selecting patients to be treated with Trastuzumab or Cetuximab in breast or colorectal cancers, respectively. Looking at the expression of immunoregulatorymolecules at the surface of tumor cells or immune infiltrates by IHC will be a requisite to treating patients with novel neutralizing immunomodulatory antibodies recognizing these proteins. Obviously, many pharmaceutical companies develop companion diagnostics together with new drugs and invest a great deal of money in identifying pharmacokinetic, toxicological and predictive biomarkers in both preclinical settings and during early and late clinical trials.

Clinical significance of minimal residual disease in young adults with standard-risk/Ph-negative precursor B-acute lymphoblastic leukemia: results of prospective study

Clinical risk classification is inaccurate in predicting outcome in adult patients with acute lymphoblastic leukemia (ALL), sometimes resulting in patients receiving inappropriate chemotherapy or stem cell transplantation. To identify complementary markers suitable for further treatment stratification in patients with standard-risk (SR)/philadelphia-negative (Ph-negative) precursor B-ALL, we evaluated the predictive value of minimal residual disease (MRD) after induction and consolidation chemotherapy in strictly defined SR/Ph-negative precursor B-ALL patients who were treated with a standard protocol using quantitative real-time polymerase chain reaction with the rearranged immunoglobulin heavy chain gene as a molecular marker. The cytologic complete response (CR) rate was 92.3 % after induction. At this time point, the molecular CR rate was 73.9%. Patients with molecular CR (MolCR) after induction had a significantly higher probability of disease-free survival (DFS; 78.8 vs 30.8%; P = .001) and of overall survival (OS; 82.4 vs 41.7%; P < .0001) compared to patients with molecular failure (MolFail). MRD at end consolidation had the same significance. Quantitative MRD assessment identified patients with MolFail after induction and/or consolidation as a high-risk group, with 3-year DFS and OS rates of 28.6 and 35.7%, respectively. Patients with MolCR after induction and consolidation were classified as low-risk and had 3-year DFS rate of 89.7% and OS rate of 93.3%. Thus, MRD quantification during treatment identified prognostic subgroups within the otherwise homogeneous SR/Ph-negative precursor B-ALL population who may benefit from individualized treatment.

Development of Hong Kong Liver Cancer Staging System With Treatment Stratification for Patients With Hepatocellular Carcinoma

We aimed to develop a prognostic classification scheme with treatment guidance for Asian patients with hepatocellular carcinoma (HCC). We collected data from 3856 patients with HCC predominantly related to hepatitis B treated at Queen Mary Hospital in Hong Kong from January 1995 through December 2008. Data on patient performance status, Child-Pugh grade, tumor status (size, number of nodules, and presence of intrahepatic vascular invasion), and presence of extrahepatic vascular invasion or metastasis were included, and randomly separated into training and test sets for analysis. Cox regression and classification and regression tree analyses were used to account for the relative effects of factors in predicting overall survival times and to classify disparate treatment decision rules, respectively; the staging system and treatment recommendation then were constructed by integration of clinical judgments. The Hong Kong Liver Cancer (HKLC) classification was compared with the Barcelona Clinic Liver Cancer (BCLC) classification in terms of discriminatory ability and effectiveness of treatment recommendation. The HKLC system had significantly better ability than the BCLC system to distinguish between patients with specific overall survival times (area under the receiver operating characteristic curve values, approximately 0.84 vs 0.80; concordance index, 0.74 vs 0.70). More importantly, HKLC identified subsets of BCLC intermediate- and advanced-stage patients for more aggressive treatments than what were recommended by the BCLC system, which improved survival outcomes. Of BCLC-B patients classified as HKLC-II in our system, the survival benefit of radical therapies, compared with transarterial chemoembolization, was substantial (5-year survival probability, 52.1% vs 18.7%; P < .0001). In BCLC-C patients classified as HKLC-II, the survival benefit of radical therapies compared with systemic therapy was even more pronounced (5-year survival probability, 48.6% vs 0.0%; P < .0001). We collected data from patients with HCC in Hong Kong to create a system to identify patients who are suitable for more aggressive treatment than the currently used BCLC system. The HKLC system should be validated in non-Asian patient populations and in patients with different etiologies of HCC.