Biomarkers Of Response Research Articles

Study of blood eosinophils and plasma periostin as biomarkers for response of COPD patients to ICS/LABA treatment

BackgroundEosinophilic airway inflammation has been detected in up to 40% of chronic obstructive pulmonary disease (COPD) patients during stable periods of the disease, and increased sputum eosinophil count was associated with better lung function, more future exacerbations, and symptoms that responded better to treatment with inhaled and oral corticosteroids.The aim of this work was to investigate the relationship of blood eosinophils and plasma periostin with lung function changes related to ICS and long-acting beta2-agonist combination treatment in stable COPD patients for 3 months.MethodsThis prospective experimental study was carried out on 50 COPD patients with post-bronchodilator FEV1/FVC ratio < 70%. Patients collected from outpatient clinics of Chest Department Tanta University Hospitals, Tanta Chest Hospital, and Mansoura Chest Hospital from February 2020 to February 2022. Patients were subjected to complete history taking, and clinical examinations including general and local examinations and laboratory investigations (complete blood count to assess eosinophilic count, arterial blood gases, and plasma periostin) and spirometry were done for all patients pre- and post-treatment. All patients received 3 months of treatment with a fixed dose of combined inhalers of ICS and LABA, and then they were classified into FEV1 responder and FEV1 non-responder according to improvement in FEV1 at least 12% and 200 mL from baseline of 3 months of combined treatment with ICS/LABA.ResultsBlood eosinophil count had a significant positive correlation with smoking index (pack/year), negative correlations with FEV1% of predicted, FEV1 actual value (L), and FEV1/FVC ratio. Plasma periostin concentration had significant positive correlation with blood eosinophil count, COPD grade, and FVC actual value (L) and significant negative correlations with FEV1% of predicted, FEV1 actual value (L), and FEV1/FVC. Cutoff values of blood eosinophil count > 265 cell/µL, plasma periostin concentration > 15.747 ng/mL, and FEV1% < 40.5% were associated with posttreatment FEV1 response.ConclusionsCOPD patients with poor lung functions regarding FEV1/FVC ratio, FEV1 actual value (L), and FEV1% of predicted as well as high blood eosinophil count and high plasma periostin concentration are predicted to have FEV1 response with fixed-dose ICS/LABA combination treatment.

DNA methylation of serotonin genes as predictive biomarkers of antidepressant treatment response

Selective serotonin reuptake inhibitors (SSRI) are frequently ineffective in treating depressive episodes and biomarkers are needed to optimize antidepressant treatment outcomes. DNA methylation levels of serotonin transporter (SLC6A4) and tryptophan hydroxylase 2 genes (TPH2) have been suggested to predict antidepressant clinical outcomes but their applicability remains uncertain. In this study, we: 1) evaluated SLC6A4/TPH2 methylation biomarker potential for predicting clinical outcomes after escitalopram treatment; 2) evaluated whether changes in SLC6A4/TPH2 methylation are informative of treatment mechanisms. We used a cohort of 90 unmedicated patients with major depressive disorder that were part of a 12-week open-label longitudinal trial and compared our observations with previous findings. Depressive symptoms were measured at baseline and after 8 and 12 weeks of treatment using the Hamilton Depression Rating Scale (HAMD6/17). We found an association between baseline TPH2 methylation and both clinical response (β:3.43; p = 0.01; 95 % CI:[0.80; 6.06]) and change in depressive symptoms after 8 weeks (β:−45.44; p = 0.01; 95 %CI:[− −78.58; −12.30]). However, we found no evidence for predictive value of any gene (TPH2 AUC: 0.74 95 % CI:[0.42;0.79]; SLC6A4: AUC: 0.61; 95 % CI: [0.48–0.78]). Methylation levels changed at the trend level for CpG sites of SLC6A4 and TPH2 over the course of 12 weeks of treatment. In addition, similar to previous observations, we found a trend for an association between methylation of SLC6A4 CpG2 (chr17:30,236,083) and HAMD17 change after 12 weeks. Our findings suggest that although TPH2 and SLC6A4 methylation may be informative of antidepressant treatment outcome, they are unlikely to prove useful as clinical predictor tools.

Phase II Trial of Gemcitabine and nab-Paclitaxel for Recurrent Osteosarcoma with Serial Monitoring Using Liquid Biopsy: A Report from the National Pediatric Cancer Foundation.

The combination of gemcitabine and docetaxel is often used to treat patients with recurrent osteosarcoma. Nab-paclitaxel has preclinical activity against osteosarcoma and is potentially less myelosuppressive than docetaxel. We conducted a prospective multi-institutional phase II trial combining gemcitabine and nab-paclitaxel for patients 12-30 years with recurrent osteosarcoma and measurable disease. A Simon's two-stage design was used to test a 4-month progression-free survival (PFS-4) of 10% vs. 35%. Patients received nab-paclitaxel 125 mg/m2 and gemcitabine 1000 mg/m2 weekly x 3 in 4-week cycles. Immunohistochemical analysis of archival tissue and serial assessment of circulating tumor cells (CTC) and circulating tumor DNA (ctDNA) using ultralow passage whole-genome sequencing were performed to identify potential biomarkers of response. Eighteen patients received 56 total cycles (median 2, range 1 - 12). Two patients (11%) experienced confirmed partial response, and 6 (33%) received > 2 cycles. The PFS-4 was 28% (95% CI 13-59%). Six patients required dose reductions and three patients were removed due to toxicities. All 18 patients had detectable CTCs, and 10 had ctDNA identified. All 8 patients with MYC amplification at study-entry experienced disease progression. Gemcitabine and nab-paclitaxel demonstrated similar clinical activity and toxicity compared to previous retrospective reports utilizing gemcitabine and docetaxel in patients with recurrent osteosarcoma. Serial analysis of CTC and ctDNA was feasible in this prospective multi-institution study and provides preliminary data on the use of these assays in patients with relapsed disease.

The baseline hemoglobin level is a positive biomarker for immunotherapy response and can improve the predictability of tumor mutation burden for immunotherapy response in cancer.

Because only a subset of cancer patients can benefit from immunotherapy, identifying predictive biomarkers of ICI therapy response is of utmost importance. We analyzed the association between hemoglobin (HGB) levels and clinical outcomes in 1,479 ICIs-treated patients across 16 cancer types. We explored the dose-dependent associations between HGB levels and survival and immunotherapy response using the spline-based cox regression analysis. Furthermore, we investigated the associations across subgroups of patients with different clinicopathological characteristics, treatment programs and cancer types using the bootstrap resampling method. HGB levels correlated positively with clinical outcomes in cancer patients receiving immunotherapy but not in those without immunotherapy. Moreover, this association was independent of other clinicopathological characteristics (such as sex, age, tumor stage and tumor mutation burden (TMB)), treatment program and cancer type. Also, this association was independent of the established biomarkers of immunotherapy response, including TMB, PD-L1 expression and microsatellite instability. The combination of TMB and HGB level are more powerful in predicting immunotherapy response than TMB alone. Multi-omics analysis showed that HGB levels correlated positively with antitumor immune signatures and negatively with tumor properties directing antitumor immunosuppression, such as homologous recombination defect, stemness and intratumor heterogeneity. The HGB measure has the potential clinical value as a novel biomarker of immunotherapy response that is easily accessible from clinically routine examination. The combination of TMB and HGB measures have better predictive performance for immunotherapy response than TMB.

Neurofilament heavy chain and chitinase 3-like 1 as markers for monitoring therapeutic response in multiple sclerosis

AimsThe aim of this study was to evaluate the association of serum neurofilament heavy chain (sNfH) and chitinase 3-like 1 (sCHI3L1) with treatment response and disease activity in multiple sclerosis (MS). MethodsThis single-center, prospective, observational cohort study was conducted at the MS Centre, University Hospital Ostrava, Czech Republic, from May 2020 to August 2023. sNfH and sCHI3L1 were determined using ELISA. A mixed-effects linear model with a log-transformed outcome variable was applied. ResultsWe analyzed 459 samples from 57 people with MS. Patients were sampled an average of 8.05 times during 21.9 months of follow-up. Those experiencing a relapse at sampling had a sNfH concentration 50 % higher than those in remission (exp(β) 1.5, 95 % CI 1.15–1.96). A longer duration of treatment was associated with lower sNfH (exp(β) 0.95, 95 % CI 0.94–0.96). Patients switched from low- to high-efficacy disease-modifying therapies (DMTs) had higher sNfH than patients treated with low-efficacy DMTs only (exp(β) 1.95, 95 % CI 1.35–2.81). Higher sCHI3L1 was associated with older age (exp(β) 1.01, 95 % CI 1.00–1.02) and longer DMT use (exp(β) 1.01, 95 % CI 1.00–1.02). sCHI3L1 values were not associated with relapse at the time of sampling, renal function, sex, or type of DMT. ConclusionIn contrast to sCHI3L1, sNfH may be a potential biomarker for monitoring treatment response and confirming clinical relapse in MS. Further research is needed to determine the long-term dynamics of sNfH and develop related treatment strategies.

The branched N-glycan of PD-L1 predicts immunotherapy responses in patients with recurrent/metastatic HNSCC

Immunotherapy has revolutionized cancer treatment, but the lack of a reliable predictive biomarker for treatment response remains a challenge. Alpha-1,6-Mannosylglycoprotein 6-β-N-Acetylglucosaminyltransferase 5 (MGAT5) is a key regulator of complex N-glycan synthesis, and its dysregulation is associated with cancer progression. The lectin Phaseolus vulgaris leukoagglutinin (PHA-L) specifically binds to mature MGAT5 products. Previous studies have indicated elevated PHA-L staining in head and neck squamous cell carcinoma (HNSCC), which implies increased activity of MGAT5. However, the specific role of MGAT5 in HNSCC remains unclear. In this study, we found significantly higher PHA-L staining and MGAT5 expression in HNSCC tumors compared to adjacent non-tumor tissues. Using a mass spectrometry (MS)-based glycoproteomic approach, we identified 163 potential protein substrates of MGAT5. Functional analysis revealed that protein substrates of MGAT5 regulated pathways related to T cell proliferation and activation. We further discovered that PD-L1 was among the protein substrates of MGAT5, and the expression of MGAT5 protected tumor cells from cytotoxic T lymphocyte (CTL) killing. Treatment of nivolumab alleviated the protective effects of MGAT5 on CTL activity. Consistently, patients with MGAT5-positive tumors showed improved responses to immunotherapy compared to those with MGAT5-negative tumors. Using purified PD-L1 from HNSCC cells and a glycoproteomic approach, we further deciphered that the N35 and N200 sites carry the majority of complex N-glycans on PD-L1. Our findings highlight the critical role of MGAT5-mediated branched N-glycans on PD-L1 in modulating the interaction with the immune checkpoint receptor PD-1. Consequently, we propose that MGAT5 could serve as a biomarker to predict patients’ responses to anti-PD-1 therapy. Furthermore, targeting the branched N-glycans at N35 and N200 of PD-L1 may lead to the development of novel diagnostic and therapeutic approaches.

A-165 Value Of Monocyte Distribution Width In Bacteremia Assessment In Emergency Department Patients

Abstract Background Monocyte distribution width (MDW) is an immune response biomarker and part of a CBC differential. MDW is derived from the distribution of monocyte volumes and elevated MDW values correlate with severe infections and sepsis in Emergency Department (ED) adult patients. Neutrophil to lymphocyte ratio (NLR) is predictive of infections and inflammatory stress. Evaluating these biomarkers could be valuable in bacteremia risk assessment. Bacteremia is the presence of viable bacteria in the bloodstream and is present in up to 20% of septic patients. Blood cultures establish pathogen identity but require culture time, potentially delaying appropriate treatment. Our goal was to characterize MDW and NLR clinical performance in ED patients with suspected bacteremia. Methods This was an observational cohort study of ED patients 18 years and older with CBC differential and blood cultures obtained. MDW values ≥20 is the published cut-off for increased infection risk. Clinical literature suggests that NLR ≥3 is abnormal. We assessed the diagnostic performance of MDW and NLR for positive blood cultures using SPSS Version 25. Results From 07/2021-09/2023, 9,400 blood cultures were ordered and 5174 patients had differential CBCs. The overall blood culture positivity rate was 14.4%. MDW and NLR performance for positive blood cultures is shown in Table 1. Conclusions Our analysis provides evidence that MDW and NLR may be useful for early recognition of bacteremia in ED patients. CBC differential results are reported sooner than culture results. MDW is directly reported, NLR can be readily calculated from the differential. Therefore both could contribute to earlier risk stratification and our findings suggest potentially distinct information from MDW and NLR. Combining MDW with other CBC parameters may provide additional diagnostic advantage. Further analysis will incorporate added clinical data for enhanced decision support in interpreting MDW values in the context of pending blood culture results.

Effects of α-olefin sulfonate (AOS) on Tubifex tubifex: toxicodynamic-toxicokinetic inferences from the general unified threshold (GUTS) model, biomarker responses and molecular docking predictions.

We investigated the potential ecological risks and harm to aquatic organisms posed by anionic surfactants such as α-olefin sulfonate (AOS), which are commonly found in industrial and consumer products, including detergents. This study assessed acute (96-h) and subchronic (14-day) responses using antioxidant activity, protein levels, and histopathological changes in Tubifex tubifex exposed to different AOS concentrations (10% of the LC50, 20% of the LC50, and a control). Molecular docking was used to investigate the potential interactions between the key stress biomarker enzymes (superoxide dismutase, catalase, and cytochrome c oxidase) of Tubifex tubifex. Acute AOS exposure showed a concentration-dependent decrease in survival, and the general unified threshold (GUTS) model revealed that survivorship is linked to individual response patterns rather than random (stochastic) fluctuations. The GUTS model also revealed dose-dependent toxicity patterns in Tubifex tubifex exposed to α-olefin sulfonate (AOS), with adaptive mechanisms at lower concentrations but significant increases in mortality beyond a certain threshold, emphasizing the role of the AOS concentration in shaping its toxicological impact. Exposure to AOS disrupted antioxidant activity, inducing oxidative stress, with GST and GPx showing positive associations with surfactant concentration and increased lipid peroxidation (elevated MDA levels); moreover, AOS exposure decreased protein concentration, signifying disturbances in vital cellular processes. Histopathological examinations revealed various tissue-level alterations, including cellular vacuolation, cytoplasmic swelling, inflammation, necrosis, and apoptosis. Molecular docking analysis demonstrated interactions between AOS and enzymes (-catalase, superoxide dismutase, and cytochrome c oxidase) in Tubifex tubifex, including hydrophobic and hydrogen bond interactions, with the potential to disrupt enzyme structures and activities, leading to cellular process disruptions, oxidative stress, and tissue damage. According to the species sensitivity distribution (SSD), the difference in toxicity between Tilapia melanopleura (higher sensitivity) and Daphnia magna (low sensitivity) to AOS suggests distinct toxicokinetic and toxicodynamic mechanisms attributable to more complex physiology in Tilapia and efficient detoxification in Daphnia due to its smaller size.

Does temperature influence on biomarker responses to copper exposure? The invasive bivalve Limnoperna fortunei (Dunker 1857) as a model

Biomarkers are useful tools for assessing the early warning effects of pollutants. However, their responses can be influenced by confounding factors. In this study, we investigated the influence of temperature on multiple biomarkers in the invasive freshwater bivalve Limnoperna fortunei exposed to copper (Cu). The mussels were exposed to low and high environmental Cu concentrations at two temperatures (15 °C and 25 °C). After 96 h, the oxidative stress, neurotoxicity, and metabolic parameters were assessed. Our results showed that temperature is a key factor influencing biomarker responses in mussels, with higher glutathione S-transferase activity and lower energy reserves at cold temperature. In addition, the effects of Cu were greater at the highest concentration at 15 °C (increased lipid peroxidation and cholinesterase activity). Overall, these findings suggest that cold stress increases the susceptibility of L. fortunei to metal effects and highlight the importance of including temperature in toxicity testing and biomonitoring. In addition, using the invasive bivalve L. fortunei as a model could prove valuable in its role as a sentinel species for other organisms.

Biomarker responses in fish caged in a rice field during a bifenthrin application

The use of pesticides in integrated rice-fish farming could have an impact on fish health. The present study aimed to evaluate, for the first time, the biological effects of the insecticide bifenthrin on fish (Piaractus mesopotamicus and Hoplosternum littorale) using a caging experiment. Fish were divided into two sites: control (C) and bifenthrin exposure (BF). Two cages (n = 8 fish/cage) per species were placed separately at each site. The BF application (Seizer ®) was carried out with a coastal sprayer according to the BF recommended dose for rice cultivation (0.1 L/ha). After 72 hours, fish were collected, and gills, liver, brain, and muscle were dissected for the analysis of biomarkers of accumulation, oxidative stress, and neurotoxicity. In P. mesopotamicus, the main changes were observed in the muscle, where BF accumulated and induced neurotoxicity (inhibition of cholinesterase activity) and oxidative stress (activation of antioxidant enzymes, decreased glutathione levels, increased lipid peroxidation). The gills and liver also showed changes in some markers of the antioxidant system. In H. littorale, BF exposure induced changes in oxidative stress biomarkers in liver (activation of antioxidant enzymes and lipid peroxidation) and gill tissues (alteration in antioxidant markers). These results show that the use of bifenthrin in rice fields poses a risk to fish farming under current pesticide management practices. Furthermore, its use could affect other species in these agroecosystems, highlighting the need for further studies to assess the ecological and productive consequences in a context of increasing pyrethroid use worldwide.

138 (PB126): A novel selective USP1 inhibitor coupled with a novel response biomarker

138 (PB126): A novel selective USP1 inhibitor coupled with a novel response biomarker

141 (PB129): Discovery of a novel KAT6A/B inhibitor with anti-tumor activity coupled with novel response biomarkers

141 (PB129): Discovery of a novel KAT6A/B inhibitor with anti-tumor activity coupled with novel response biomarkers

Artificial Intelligence Applications in Oral Cancer and Oral Dysplasia.

Oral squamous cell carcinoma (OSCC) is a highly unpredictable disease with devastating mortality rates that have not changed over the past decades, in the face of advancements in treatments and biomarkers, which have improved survival for other cancers. Delays in diagnosis are frequent, leading to more disfiguring treatments and poor outcomes for patients. The clinical challenge lies in identifying those patients at the highest risk of developing OSCC. Oral epithelial dysplasia (OED) is a precursor of OSCC with highly variable behavior across patients. There is no reliable clinical, pathological, histological, or molecular biomarker to determine individual risk in OED patients. Similarly, there are no robust biomarkers to predict treatment outcomes or mortality in OSCC patients. This review aims to highlight advancements in artificial intelligence (AI)-based methods to develop predictive biomarkers of OED transformation to OSCC or predictive biomarkers of OSCC mortality and treatment response. Biomarkers such as S100A7 demonstrate promising appraisal for the risk of malignant transformation of OED. Machine learning-enhanced multiplex immunohistochemistry workflows examine immune cell patterns and organization within the tumor immune microenvironment to generate outcome predictions in immunotherapy. Deep learning (DL) is an AI-based method using an extended neural network or related architecture with multiple "hidden" layers of simulated neurons to combine simple visual features into complex patterns. DL-based digital pathology is currently being developed to assess OED and OSCC outcomes. The integration of machine learning in epigenomics aims to examine the epigenetic modification of diseases and improve our ability to detect, classify, and predict outcomes associated with epigenetic marks. Collectively, these tools showcase promising advancements in discovery and technology, which may provide a potential solution to addressing the current limitations in predicting OED transformation and OSCC behavior, both of which are clinical challenges that must be addressed in order to improve OSCC survival.

NRN1 genetic variability and methylation changes as biomarkers for cognitive remediation therapy response in schizophrenia

Cognitive remediation therapy (CRT) demonstrates potential in enhancing cognitive function in schizophrenia (SZ), though the identification of molecular biomarkers remains challenging. The Neuritin-1 gene (NRN1) emerges as a promising candidate gene due to its association with SZ, cognitive performance and response to neurotherapeutic treatments. We aimed to investigate whether NRN1 genetic variability and methylation changes following CRT are related to cognitive improvements.Twenty-five SZ patients were randomly assigned to CRT or treatment-as-usual (TAU) groups, with cognitive function and NRN1 methylation assessed pre- and post-intervention using the MATRICS Consensus Cognitive Battery and EpiTYPER. Besides, eleven NRN1 polymorphisms were genotyped. Methylation changes (Δm = post - pre) were analyzed via sparse Partial Least Square Discriminant Analysis (sPLS-DA) to identify latent components (LCs) distinguishing CRT from TAU. To further explore methylation patterns of these LCs, CpG units were grouped into two subsets, yielding Δm means for those with increased and decreased methylation. Cognitive changes (Δcog = post - pre) were used to identify CRT improvers (CRTI, Δcog ≥1), and the association between methylation changes and cognitive improvements post-therapy was also tested.We identified two LCs that differentiated CRT from TAU with a classification error rate of 0.28. The main component, LC1, included 25 CpG units. The subsets of CpG units with increased and decreased post-therapy methylation differed significantly between the two treatment arms, suggesting that differences were not merely data-driven but reflected meaningful biological variation. Additionally, CpG units linked to therapy were also associated with cognitive improvement, with LC1 and the subset of CpG units showing increased methylation post-therapy distinguishing CRT-I from the rest of the patients across multiple cognitive domains. Furthermore, the effect of LC1 on speed processing improvement after CRT was enhanced by considering the NRN1-rs9405890 polymorphism. Notably, these CpG units, particularly those with increased methylation after CRT, overlapped with key gene regulatory elements.Our model, integrating genetics and epigenetics, boosts the understanding of CRT response variability and highlights this multi-level approach as a promising strategy for identifying potential NRN1-related biomarkers of CRT effects, though further studies with larger samples are needed.

Cancer-Associated Fibroblasts Expressing Sulfatase 1 Facilitate VEGFA-Dependent Microenvironmental Remodeling to Support Colorectal Cancer.

Tumor stroma plays a critical role in fostering tumor progression and metastasis. Cancer-associated fibroblasts (CAF) are a major component of the tumor stroma. Identifying the key molecular determinants for the protumor properties of CAFs could enable the development of more effective treatment strategies. In this study, through analyses of single-cell sequencing data, we identified a population of CAFs expressing high levels of sulfatase 1 (SULF1), which was associated with poor prognosis in patients with colorectal cancer. Colorectal cancer models using mice with conditional SULF1 knockout in fibroblasts revealed the tumor-supportive function of SULF1+ CAFs. Mechanistically, SULF1+ CAFs enhanced the release of VEGFA from heparan sulfate proteoglycan. The increased bioavailability of VEGFA initiated the deposition of extracellular matrix and enhanced angiogenesis. In addition, intestinal microbiota-produced butyrate suppressed SULF1 expression in CAFs through its histone deacetylase (HDAC) inhibitory activity. The insufficient butyrate production in patients with colorectal cancer increased the abundance of SULF1+ CAFs, thereby promoting tumor progression. Importantly, tumor growth inhibition by HDAC was dependent on SULF1 expression in CAFs, and patients with colorectal cancer with more SULF1+ CAFs were more responsive to treatment with the HDAC inhibitor chidamide. Collectively, these findings unveil the critical role of SULF1+ CAFs in colorectal cancer and provide a strategy to stratify patients with colorectal cancer for HDAC inhibitor treatment. Significance: SULF1+ cancer-associated fibroblasts play a tumor-promoting role in colorectal cancer by stimulating extracellular matrix deposition and angiogenesis and can serve as a biomarker for the therapeutic response to HDAC inhibitors in patients.

175P Thrombospondin-4 as potential cerebrospinal fluid biomarker for therapy response in pediatric 5q-associated spinal muscular atrophy patients

175P Thrombospondin-4 as potential cerebrospinal fluid biomarker for therapy response in pediatric 5q-associated spinal muscular atrophy patients

432 (PB420): A multi-omics approach using transcriptomic and spatial analysis to identify predictive immune biomarkers of response and toxicity following chemotherapy in head and neck cancers

432 (PB420): A multi-omics approach using transcriptomic and spatial analysis to identify predictive immune biomarkers of response and toxicity following chemotherapy in head and neck cancers

322 (PB310): A multi-omics approach to identify biomarkers of response to the novel and selective SRC/YES1 inhibitor NXP900

322 (PB310): A multi-omics approach to identify biomarkers of response to the novel and selective SRC/YES1 inhibitor NXP900

79 (PB067): Identification of biomarkers of response and the mechanism of action of a selective androgen receptor modulator in estrogen receptorpositive breast cancer patient-derived xenografts

79 (PB067): Identification of biomarkers of response and the mechanism of action of a selective androgen receptor modulator in estrogen receptorpositive breast cancer patient-derived xenografts

P2.11A.04 Exhaled Breath Biomarkers of Response to Immunotherapy in Advanced Non-Small Lung Cancer

P2.11A.04 Exhaled Breath Biomarkers of Response to Immunotherapy in Advanced Non-Small Lung Cancer