Mohammad Abu Zaid

Plasma biomarkers of risk for death in a multicenter phase 3 trial with uniform transplant characteristics post-allogeneic HCT

A phase 3 clinical trial (BMT CTN 0402) comparing tacrolimus/sirolimus (Tac/Sir) vs tacrolimus/methotrexate (Tac/Mtx) as graft-versus-host disease (GVHD) prophylaxis after matched-related allogeneic hematopoietic cell transplantation (HCT) recently showed no difference between study arms in acute GVHD-free survival. Within this setting of a prospective, multicenter study with uniform GVHD prophylaxis, conditioning regimen, and donor source, we explored the correlation of 10 previously identified biomarkers with clinical outcomes after allogeneic HCT. We measured biomarkers from plasma samples collected in 211 patients using enzyme-linked immunosorbent assay (Tac/Sir = 104, Tac/Mtx = 107). High suppression of tumorigenicity-2 (ST2) and T-cell immunoglobulin mucin-3 (TIM3) at day 28 correlated with 2-year nonrelapse mortality in multivariate analysis (P = .0050, P = .0075, respectively) and in a proportional hazards model with time-dependent covariates (adjusted hazard ratio: 2.43 [1.49-3.95], P = .0038 and 4.87 [2.53-9.34], P < .0001, respectively). High ST2 and TIM3 correlated with overall survival. Chemokine (C-X-C motif) ligand 9 (CXCL9) levels above the median were associated with chronic GVHD compared with levels below the median in a time-dependent proportional hazard analysis (P = .0069). Low L-Ficolin was associated with hepatic veno-occlusive disease (P = .0053, AUC = 0.80). We confirmed the correlation of plasma-derived proteins, previously assessed in single-center cohorts, with clinical outcomes after allogeneic HCT within this prospective, multicenter study.

Targeted serum metabolite profiling and sequential metabolite ratio analysis for colorectal cancer progression monitoring

Colorectal cancer (CRC) is one of the most prevalent cancers worldwide and a major cause of human morbidity and mortality. In addition to early detection, close monitoring of disease progression in CRC can be critical for patient prognosis and treatment decisions. Efforts have been made to develop new methods for improved early detection and patient monitoring; however, research focused on CRC surveillance for treatment response and disease recurrence using metabolomics has yet to be reported. In this proof of concept study, we applied a targeted liquid chromatography tandem mass spectrometry (LC-MS/MS) metabolic profiling approach focused on sequential metabolite ratio analysis of serial serum samples to monitor disease progression from 20 CRC patients. The use of serial samples reduces patient to patient metabolic variability. A partial least squares-discriminant analysis (PLS-DA) model using a panel of five metabolites (succinate, N2, N2-dimethylguanosine, adenine, citraconic acid, and 1-methylguanosine) was established, and excellent model performance (sensitivity = 0.83, specificity = 0.94, area under the receiver operator characteristic curve (AUROC) = 0.91 was obtained, which is superior to the traditional CRC monitoring marker carcinoembryonic antigen (sensitivity = 0.75, specificity = 0.76, AUROC = 0.80). Monte Carlo cross validation was applied, and the robustness of our model was clearly observed by the separation of true classification models from the random permutation models. Our results suggest the potential utility of metabolic profiling for CRC disease monitoring.

Exploring Metabolic Profile Differences between Colorectal Polyp Patients and Controls Using Seemingly Unrelated Regression

Despite the fact that colorectal cancer (CRC) is one of the most prevalent and deadly cancers in the world, the development of improved and robust biomarkers to enable screening, surveillance, and therapy monitoring of CRC continues to be evasive. In particular, patients with colon polyps are at higher risk of developing colon cancer; however, noninvasive methods to identify these patients suffer from poor performance. In consideration of the challenges involved in identifying metabolite biomarkers in individuals with high risk for colon cancer, we have investigated NMR-based metabolite profiling in combination with numerous demographic parameters to investigate the ability of serum metabolites to differentiate polyp patients from healthy subjects. We also investigated the effect of disease risk on different groups of biologically related metabolites. A powerful statistical approach, seemingly unrelated regression (SUR), was used to model the correlated levels of metabolites in the same biological group. The metabolites were found to be significantly affected by demographic covariates such as gender, BMI, BMI(2), and smoking status. After accounting for the effects of the confounding factors, we then investigated potential of metabolites from serum to differentiate patients with polyps and age matched healthy controls. Our results showed that while only valine was slightly associated, individually, with polyp patients, a number of biologically related groups of metabolites were significantly associated with polyps. These results may explain some of the challenges and promise a novel avenue for future metabolite profiling methodologies.

CXCL10: most consistent cGVHD biomarker?

In this issue of Blood , Kariminia et al report that serum CXCL10 is the only consistent biomarker for chronic graft-versus-host disease (cGVHD) and is associated with low peripheral blood levels of CXCR3 + natural killer (NK) cells. 1

Predicting Cardiovascular Disease Among Testicular Cancer Survivors After Modern Cisplatin-based Chemotherapy: Application of the Framingham Risk Score

Micro‐Abstract Testicular cancer survivors are at increased risk of cardiovascular disease after cisplatin‐based chemotherapy. Among 787 testicular cancer survivors, the Framingham Risk Score for cardiovascular disease was elevated among less educated and less vigorously active patients, but did not differ by chemotherapy regimen (4 cycles of EP [etoposide and cisplatin] or 3‐4 cycles of BEP [bleomycin, etoposide, and cisplatin]). Follow‐up and counseling in high‐risk subgroups is recommended. Background: Testicular cancer survivors (TCSs) are at increased risk of cardiovascular disease (CVD) after cisplatin‐based chemotherapy (CBCT). Identifying at‐risk survivors would allow early intervention, but risk prediction tools such as the Framingham Risk Score (FRS) have not been applied to TCSs given modern chemotherapy. Methods: TCSs > 1 year post‐CBCT were evaluated. Associations between FRS and clinical, socioeconomic, and lifestyle measures and treatment regimen (4 cycles, etoposide and cisplatin [EP × 4]); 3 or 4 cycles, bleomycin plus EP (BEP × 3, BEP × 4) were analyzed with general linear multivariable models. Controls from the National Health and Nutrition Examination Survey were matched 1:1 to TCSs by age, race, and education with differences in mean FRS evaluated with 2‐sided t tests. Results: Of 787 TCSs (median age, 37.3 years; median follow‐up, 4.2 years), 284, 342, and 161 received EP × 4, BEP × 3, or BEP × 4, respectively. TCSs had higher median systolic blood pressure (126 vs. 119 mm Hg; P < .001), but fewer were smokers (8.4% vs. 28.2%; P < .001) than controls. In multivariable analysis, no significant differences in FRS between EP × 4, BEP × 3, and BEP × 4 were observed, but less than college education (P < .001) and lack of vigorous exercise (P = .006) were associated with higher FRS. Mean FRS did not differ between TCSs and controls (6.8% vs. 7.3%; P = .67). Conclusion: This is the first study to apply the office‐based FRS to TCSs. Chemotherapy regimen (BEP × 3 vs. EP × 4) was not associated with FRS, but less educated and less vigorously active patients had higher FRS, and present a high‐risk subgroup for intense follow‐up and counseling.

Altered metabolite levels and correlations in patients with colorectal cancer and polyps detected using seemingly unrelated regression analysis

IntroductionMetabolomics technologies enable the identification of putative biomarkers for numerous diseases; however, the influence of confounding factors on metabolite levels poses a major challenge in moving forward with such metabolites for pre-clinical or clinical applications.ObjectivesTo address this challenge, we analyzed metabolomics data from a colorectal cancer (CRC) study, and used seemingly unrelated regression (SUR) to account for the effects of confounding factors including gender, BMI, age, alcohol use, and smoking.MethodsA SUR model based on 113 serum metabolites quantified using targeted mass spectrometry, identified 20 metabolites that differentiated CRC patients (n = 36), patients with polyp (n = 39), and healthy subjects (n = 83). Models built using different groups of biologically related metabolites achieved improved differentiation and were significant for 26 out of 29 groups. Furthermore, the networks of correlated metabolites constructed for all groups of metabolites using the ParCorA algorithm, before or after application of the SUR model, showed significant alterations for CRC and polyp patients relative to healthy controls.ResultsThe results showed that demographic covariates, such as gender, BMI, BMI2, and smoking status, exhibit significant confounding effects on metabolite levels, which can be modeled effectively.ConclusionThese results not only provide new insights into addressing the major issue of confounding effects in metabolomics analysis, but also shed light on issues related to establishing reliable biomarkers and the biological connections between them in a complex disease.

508. IL-33/ST2 Triggering of IL-9-Secreting T Cells: From Proteomics to Therapeutics

As one of the most validated immunotherapies to date, allogeneic hematopoietic cell transplantation (allo-HCT) is a potentially curative option for high-risk hematological malignancies, particularly acute myeloid leukemia (AML). The immunotherapeutic activity of allo-HCT is known as the graft-vs-leukemia (GVL) activity. However, GVL activity is often accompanied by T-cell reactivity to allo-antigens in normal host tissues, which leads to graft-versus-host disease (GVHD), another major cause of death after HCT. Therefore, there is a great unmet need to improve the current process of allo-HCT through increasing the GVL activity and decreasing GVHD. We have shown that an elevated plasma level of soluble (s)ST2 in HCT patients is a risk factor for severe GVHD. ST2 blockade reduces sST2-producing T cells while maintaining protective membrane (m)ST2-expressing T cells such as type 2 T cells and regulatory T cells during aGVHD. A novel IL-9 producing T helper subset, Th9, expresses mST2. Furthermore, Th9 cells and IL-9 producing CD8 cytotoxic (Tc9) cells have higher antitumor activity than Th1 and Tc1 cells in melanoma models. Interestingly, we found that the addition of IL-33 during T9 differentiation (T9IL-33) increased expression of mST2 and PU.1, a transcription factor that promotes IL-9 production in both CD4 and CD8 T cells. Adoptive transfer of T9IL-33 cells with bone marrow cells in a murine model of HCT resulted in less severe GVHD compared to transfer of T9IL-33 cells generated from ST2−/− or IL-9−/− T cells. Furthermore, cytolytic molecules implicated in anti-leukemic activity (granzyme B and perforin) were upregulated in WT T9IL-33 cells while ST2−/− T9IL-33 cells did not. WT T9IL-33 cells also exhibited higher anti-leukemic activity when cultured with a retrovirally transduced MLL-AF9 leukemic cells in comparison to ST2−/− T9IL-33 in in vitro cytolytic assays. In vivo GVL experiments with MLL-AF9 AML and adoptive transfer of T9IL-33 cells resulted in increased survival compared to syngeneic mice, allo-HCT mice transferred with T1 cells, or T9 cells or T9IL-33 cells generated from ST2−/− or IL-9−/− T cells (Figure 1Figure 1). Human T9 cells are poorly studied. Here we demonstrate that IL-33 has the same impact on human T cells through enhancing IL-9 and Granzyme B production compared to T9 cells as well as demonstrated higher in vitro anti-leukemic cytolytic activity when incubated with MOLM14, an aggressive AML tumor cell line expressing FLT3/ITD mutations. Importantly, CD8α expression was upregulated in WT T9IL-33 (both CD4 and CD8) cells in comparison to ST2−/− T9IL-33 cells, and CD8α blockade with neutralizing antibody during allogeneic specific T9IL-33 differentiation reduced cytotoxicity of both murine T9IL-33, and human T9IL-33 cells as compared to the cell blocked with isotype control, suggesting that CD8α was associated with MHC-restricted cytolytic activity in T9IL-33 cells. Altogether, our observations demonstrated that adoptive transfer of T9IL-33 cells represents a promising cellular therapy following HCT.View Large Image | Download PowerPoint Slide

Abstract B52: Targeted LC-MS/MS metabolic profiling for colon cancer progression monitoring

Introduction: Colorectal cancer (CRC) is one of the most prevalent cancers worldwide, and a major cause of human morbidity and mortality. A number of current efforts are focused on earlier detection of colon cancer using a variety of technologies including genomics, proteomics and metabolomics. Research focused on CRC disease status surveillance using metabolomics or other approaches has not been reported; Close monitoring of disease progression (DP) in CRC can be critical for patients9 prognosis management and treatment decisions. In this study we investigate a targeted LC-MS/MS approach for serum metabolic profiling to monitor and predict patient disease progression, using a panel of significantly altered metabolites as potential biomarkers. Methods: 59 serum samples from 21 CRC patients were analyzed, including 23 samples from DP patients and 36 from other CRC disease status (e.g., stable disease and complete remission). Chromatographic separations were performed via an Agilent HPLC system installed with two hydrophilic interaction chromatography (HILIC) columns, and then targeted data acquisition was performed in multiple-reaction-monitoring (MRM) mode using an AB Sciex QTrap 5500 mass spectrometer. We monitored 106 and 58 MRM transitions in negative and positive mode, respectively. Univariate and multivariate statistical analyses (such as the Mann- Whitney U-test and PLS-DA) were applied for metabolite biomarker discovery and model development on a selected set of promising biomarker candidates. Monte Carlo cross validation (MCCV) was performed to evaluate model robustness. Results and conclusion: LC-MS/MS targeted analysis provided a robust system for metabolic profiling of CRC patient disease status monitoring using serum samples. Targeted screening of 164 metabolites, representing more than 20 different classes (such as amino acids, carboxylic acids, pyridines, and etc.) and from 25 important metabolic pathways (e.g., TCA cycle, amino acid metabolism, purine and pyrimidine metabolism, and glycolysis, and etc.) was performed using both positive and negative ionization modes. 131 metabolites could be reproducibly detected in the serum samples, with an average CV of 7.1% measured in pooled serum quality control samples. After univariate analysis, 36 metabolites from different classes, such as monosaccharides, amino acids, carboxylic acids and nucleosides, showed a significant statistical difference (p Citation Format: Jiangjiang Zhu, Danijel Djukovic, Lingli Deng, Lingli Deng, Haiwei Gu, Farhan Himmati, Mohammad Abu Zaid, E. Gabriela Chiorean, E. Gabriela Chiorean, Daniel Raftery, Daniel Raftery. Targeted LC-MS/MS metabolic profiling for colon cancer progression monitoring. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr B52.

Cardiovascular disease (CVD) risk factors and health behaviors following cisplatin-based chemotherapy (CHEM): A multi-institutional study of testicular cancer survivors (TCS).

129 Background: TCS are at increased risk of CVD, but few clinical studies have comprehensively evaluated CVD risk factors through physical exams, lipid panels, and health behaviors in North American patients. METHODS Eligible TCS were < 50 y at diagnosis and treated with only first line CHEM. TCS underwent physical exams, had fasting lipid panels, and completed questionnaires regarding co-morbidities and health behaviors. Age, race, and educational status-matched controls (1:1) were chosen from the general population using the 2011-2012 National Health and Nutrition Examination Survey. Odds ratios (OR) of outcomes among TCS versus matched controls were estimated using logistic regression models. RESULTS We evaluated 680 consecutively enrolled TCS. Median age at diagnosis was 31 y (range, 15-49) and at clinical evaluation 38 y (range, 19-68). Median time since CHEM was 4.3 y (range, 1-30). Compared to normative controls, TCS were more likely to be overweight (OR = 1.65; 95% CI 1.26-2.16), have total cholesterol ≥ 240 mg/dL (OR = 2.19; 95% CI 1.12-4.28) and LDL ≥ 160 mg/dL (OR = 3.05; 95% CI 1.03-9.00), and report alcohol use > 2 days/week (OR = 2.13; 95% CI 1.64-2.77). In contrast, they were more likely to have a waist circumference < 40 inches (OR = 1.32; 95% CI 1.04-1.66); engage in vigorous (OR = 2.64; 95% CI 2.11-3.29) or moderate (OR = 1.62; 95% CI 1.30-2.03) physical activity, and not smoke (OR = 2.95; 95% CI 2.14-4.08). TCS were about 3 times more likely overall to report excellent, very good, or good health compared to controls (P < 0.05). No significant differences were found comparing HDL, triglycerides, or self-reported hypertension (P > 0.05). CONCLUSIONS Although North American TCS appear more likely to exercise and abstain from smoking compared to normative controls, a greater proportion are overweight and have higher fasting total cholesterol and LDL levels. Health care providers should screen TCS for CVD risk factors, and encourage practices consistent with a healthy lifestyle. Future research should elucidate mechanisms of increased CVD risk and ultimately develop customized prevention and intervention strategies.