Urine Immunofixation Electrophoresis for Diagnosis of Monoclonal Gammopathy: Evaluation of Methods for Urine Concentration.

BACKGROUND: Examination of urine by immunofixation electrophoresis (UIFE) is one of the tests recommended for screening and monitoring of monoclonal gammopathies, especially multiple myeloma. Unlike the serum free light chain measurement, a positive result on urine immunofixation is diagnostic for monoclonal immunoglobulin light chains. Urine is usually concentrated, generally by membrane filtration, prior to electrophoresis. METHODS: Alternative methods to membrane filtration for urine concentration were examined. Residual urine specimens submitted for urine protein electrophoresis were concentrated by precipitation of the proteins by ammonium sulfate salt precipitation, precipitation with ethanol and acetonitrile, and by desiccation. The concentrated specimens were subjected to immunofixation electrophoresis using antisera to free light chains (FLC). The results were compared with those from conventional immunofixation electrophoresis using specimens concentrated by membrane filtration. RESULTS: Ammonium sulfate, ethanol, and acetonitrile precipitation results were less than satisfactory. Concentration by desiccation provided results comparable, if not better than, those by membrane filtration and conventional UIFE. The cost of desiccation is minimal compared to more than $5.00/specimen cost of concentration by membrane filtration. The differences in the results with conventional UIFE and the method described here are likely due to (a) variability in the reactivity of different antisera to free monoclonal light chains, and (b) obscuration of monoclonal free light chains by co-migration with intact immunoglobulin monoclonal proteins. CONCLUSIONS: Concentrating urine by desiccation for immunofixation electrophoresis is technically simple, inexpensive, and provides results comparable to concentrating by membrane filtration. Using FLC provides a more sensitive assay than using conventional antisera.

Quantification of Free Immunoglobulin Light Chains in Urine.

BACKGROUND: The serum-free immunoglobulin light chain assay has been recommended as a screening test for monoclonal gammopathy. We evaluated the usefulness of urine free immunoglobulin light concentration for selection of specimens for immunofixation electrophoresis. METHODS: Using kits from The Binding Site for Freelite ®, we validated examination of urine for measuring free κ and λ light chains. The results of urine free light chain concentrations were evaluated to ascertain if the results could be used to reduce the number of specimens requiring urine protein immunofixation electrophoresis. RESULTS: In the 515 specimens examined, there was no evidence of monoclonal gammopathy or history of monoclonal gammopathy in 331. Monoclonal κ or λ light chains were detectable in 42 and 30 specimens, respectively. There was history of κ or λ chain associated monoclonal gammopathy in 62 and 50 patients, respectively. In the 38 monoclonal κ positive urine specimens, with light chain data, κ/λ ratio was >5.83 in all specimens. In 27 specimens positive for monoclonal λ light chains, with light chain data, the urine λ/κ ratio was > 0.17 in 24 of 27 specimens and > 0.041 in all specimens. In patients without monoclonal gammopathy all specimens had a κ/λ ratio of >5.83 or λ/κ ratio >0.17. CONCLUSIONS: The Freelite ® assay from The Binding Site is suitable for quantification of free light chains in urine. In patients with known history of monoclonal gammopathy, urine immunofixation electrophoresis may be omitted in specimens with κ/λ ratio of <5.83 for κ associated lesions and λ/κ ratio of <0.041 for λ associated lesions. However, the results do not support using this test for first-time urine testing for monoclonal light chains as it is not predictive of positive result, nor does it exclude a monoclonal light chain in urine.

Inhibitors of the CD73-adenosinergic checkpoint as promising combinatory agents for conventional and advanced cancer immunotherapy.

The cell surface enzyme CD73 is increasingly appreciated as a pivotal non-redundant immune checkpoint (IC) in addition to PD-1/PD-L1 and CTLA-4. CD73 produces extracellular adenosine (eADO), which not only inhibits antitumor T cell activity via the adenosine receptor (AR) A2AR, but also enhances the immune inhibitory function of cancer-associated fibroblasts and myeloid cells via A2BR. Preclinical studies show that inhibition of the CD73-adenosinergic pathway in experimental models of many solid tumors either as a monotherapy or, more effectively, in combination with PD-1/PD-L1 or CTLA-4 IC blockades, improves antitumor immunity and tumor control. Consequently, approximately 50 ongoing phase I/II clinical trials targeting the CD73-adenosinergic IC are currently listed on https://clinicaltrials.gov. Most of the listed trials employ CD73 inhibitors or anti-CD73 antibodies alone, in combination with A2AR antagonists, and/or with PD-1/PD-L1 blockade. Recent evidence suggests that the distribution of CD73, A2AR and A2BR in tumor microenvironments (TME) is heterogeneous, and this distribution affects CD73-adenosinergic IC function. The new insights have implications for the optimally effective, carefully tailored approaches to therapeutic targeting of this essential IC. In the mini-review, we briefly discuss the cellular and molecular mechanisms of CD73/eADO-mediated immunosuppression during tumor progression and therapy in the spatial context of the TME. We include preclinical data regarding therapeutic CD73-eADO blockade in tumor models as well as available clinical data from completed trials that targeted CD73-adenosinergic IC with or without PD-1/PD-L1 inhibitors and discuss factors that are potentially important for optimal therapeutic outcomes in cancer patients.

Urine Protein Immunofixation Electrophoresis: Free Light Chain Urine Immunofixation Electrophoresis Is More Sensitive than Conventional Assays for Detecting Monoclonal Light Chains and Could Serve as a Marker of Minimal Residual Disease.

BACKGROUND: Immunoglobulin monoclonal light chains (MLCs) in serum and urine are markers for monoclonal gammopathy and could serve as markers of minimal residual disease (MRD) in multiple myeloma (MM). Excretion of MLCs in urine is known to result in renal damage and shorter survival in patients with LC-predominant MM. METHODS: Retrospective review of urine immunofixation in 1738 specimens at 3 medical centers was conducted to assess the utility of urinalysis for diagnosis and monitoring of monoclonal gammopathy. We tested 228 stored urine specimens via the modified urine immunofixation method, using antisera to assay free LCs (FLCs). RESULTS: Our review of urine immunofixation results and medical records validated the theory that the only meaningful value-added finding was detection of monoclonal free light chains. Examination of 228 urine specimens using our novel method revealed 18.4% additional positive results. The rate of incremental findings for lambda LCs was nearly 3-fold higher than for kappa LCs. CONCLUSIONS: The new method of urine immunofixation is significantly more sensitive and more efficient than the conventional method for detecting MLCs in urine. The new assay appears to be sensitive enough to prove that MLCs serve as a marker of MRD in MM.

Pathology Job Search and Interview: Perspectives on the United States Experience.

CONTEXT.­: Pathology training is focused on the attainment of clinical, diagnostic, and administrative skills. Preparation for employment search and the interview process are often neglected. Given that a near majority of pathology trainees in the United States are graduates of foreign medical schools, training in the job search and interview process according to local customs, norms, and expectations has greater salience for individuals new to the United States. OBJECTIVE.­: To offer perspectives on 2 components of the job search process: (1) finding a suitable job opening in academic and private practice settings and (2) preparing for an interview. We have provided a set of common interview questions and suggested preparatory methodology. The differences in the process and expectations in academic settings and private practice operations are highlighted. Engaging in the job search process early and networking are emphasized. We have also suggested approaches for pathology teachers and mentors in guiding trainees in a job search and preparation for an interview. DATA SOURCES.­: The information and opinions expressed in this communication are based on the personal experiences of 4 senior pathologists in academic and private practice settings. CONCLUSIONS.­: Start networking early. Leverage contacts with teachers, attending pathologists, senior residents, and people at national meetings to locate appropriate job opportunities. Seek assistance from attending pathologists in preparing a curriculum vitae and cover letter. Prepare for the questions that may come up in an interview. A dress rehearsal for an interview is strongly recommended.

Serum Free Light Chain Quantification Testing: Comparison of Two Methods for Disease Monitoring.

BACKGROUND: Levels of free immunoglobulin light chains in serum and urine are a sensitive measure of dysregulated immunoglobulin synthesis. The development of an assay for free light chains in serum was a major advance in laboratory testing for monoclonal gammopathies. The original assay by The Binding Site, called Freelite®, has been in common use in laboratory monitoring of monoclonal gammopathies. Two clinical entities, myeloma-defining condition and light chain-predominant multiple myeloma, rely on quantitative measurements of serum free light chains. METHODS: Using polyclonal antisera specific to free light chains, Diazyme Laboratories developed a latex immunoturbidimetric assay for quantification of human kappa and lambda serum free light chains. We evaluated the Diazyme assay by comparing the results of kappa and lambda free light chain quantification, and kappa/lambda ratio with the results on the same specimens by the Freelite method. We also compared the correlation of the 2 methods to evaluate response to treatment and to changes in clinical status of patients with multiple myeloma. RESULTS: The results of Freelite and Diazyme methods are comparable. There was no statistically significant difference in the performance of the 2 assays for quantification of light chains, kappa/lambda ratio, or correlation of clinical parameters from patients with multiple myeloma at various stages of monitoring the disease in 2 geographically diverse laboratory and clinical environments. CONCLUSIONS: The Diazyme method is comparable to Freelite and provides an opportunity to add the test to front-end automation and improvement in efficiency of the assay.

Lipidomic Profiling Identifies a Novel Lipid Signature Associated with Ethnicity-Specific Disparity of Bladder Cancer.

Bladder Cancer (BLCA) is the ninth most frequently diagnosed cancer globally and the sixth most common cancer in the US. African Americans (AA) exhibit half the BLCA incidence compared to European Americans (EA), but they have a 70% higher risk of cancer-related death; unfortunately, this disparity in BLCA mortality remains poorly understood. In this study, we have used an ethnicity-balanced cohort for unbiased lipidomics profiling to study the changes in the lipid fingerprint for AA and EA BLCA tissues collected from similar geographical regions to determine a signature of ethnic-specific alterations. We identified 86 lipids significantly altered between self-reported AA and EA BLCA patients from Augusta University (AU) cohort. The majority of altered lipids belong to phosphatidylcholines (PCs), phosphatidylethanolamines (PEs), ly sophosphatidylcholines (lysoPCs), phosphatidylserines (PSs), and diglycerides (DGs). Interestingly, levels of four lysoPCs (lyso PCs 20:3, lyso PCs 22:1, lyso PCs 22:2, and lyso PCs 26:1) were elevated while, in contrast, the majority of the PCs were reduced in AA BLCA. Significant alterations in long-chain monounsaturated (MonoUN) and polyunsaturated (PolyUN) lipids were also observed between AA and EA BLCA tumor tissues. These first-in-field results implicate ethnic-specific lipid alterations in BLCA.

Light Chain-Predominant Multiple Myeloma Subgroup: Impaired Renal Function Correlates with Decreased Survival.

OBJECTIVE: Patients with light chain-predominant multiple myeloma have been shown to exhibit shorter survival. Retrospective comparison of clinical and laboratory data was undertaken to ascertain the likely cause(s) of this observation. METHODS: Records of patients with multiple myeloma seen at 1 institution revealed 316 patients with conventional and 71 patients with light chain-predominant multiple myelomas with secretion of intact immunoglobulins. Laboratory and clinical findings in the 2 groups were compared. RESULTS: Patients with light chain-predominant multiple myeloma had a significantly higher death rate, a higher rate of chronic dialysis, a lower estimated glomerular filtration rate and serum albumin, a significantly higher urine protein concentration, and a significantly higher prevalence of hypertension and blood transfusion requirements. Other clinical and laboratory parameters surveyed were not significantly different between the 2 groups. CONCLUSION: The shorter survival of patients with light chain-predominant multiple myeloma is clearly associated with renal damage caused by excess free immunoglobulin light chains. Renal damage may be ameliorated by early aggressive treatment with chemotherapy, plasmapheresis, and dialysis; a multi-institutional prospective controlled trial would be needed to test this hypothesis.

Multiple myeloma: Detection of free monoclonal light chains by modified immunofixation electrophoresis with antisera against free light chains.

INTRODUCTION: Neoplastic monoclonal gammopathies are frequently associated with production of excess free monoclonal light chains. A sensitive method for detecting free monoclonal light chains in serum could provide a marker for residual/minimal residual disease and as an adjunct to serum protein electrophoresis to serve as a screening method for monoclonal gammopathies. METHODS: Conventional serum immunofixation electrophoresis was modified by applying undiluted serum samples, and staining for serum free light chains with antisera specific to free light chains. Washing/blotting of gels was enhanced to remove residual proteins that did not bind to the antibodies including intact monoclonal immunoglobulins. Results from this modified immunofixation electrophoresis were compared with results from commercially available MASS-FIX/MALDI assay. RESULTS: Monoclonal free kappa light chains could be detected to a concentration of about 1.78 mg/L and monoclonal free lambda light chains to a concentration of about 1.15 mg/L without the need for special equipment. Comparison of these thresholds with parallel results from a commercially available MASS-FIX/MALDI assay revealed the modified electrophoretic method to be more sensitive in the context of free monoclonal light chains. CONCLUSIONS: Modified serum immunofixation electrophoresis has been shown to detect low levels of monoclonal free light chains at a sensitivity suitable for the method to be used in detecting minimal residual disease, and potentially in a screening assay for monoclonal gammopathies. The disparity in the results with commercially available MASS-FIX/MALDI assay is postulated to be due to limited repertoire of reactivity of nanobodies of camelid origin.

Inhibition of the BTK-IDO-mTOR axis promotes differentiation of monocyte-lineage dendritic cells and enhances anti-tumor T cell immunity.

Monocytic-lineage inflammatory Ly6c+CD103+ dendritic cells (DCs) promote antitumor immunity, but these DCs are infrequent in tumors, even upon chemotherapy. Here, we examined how targeting pathways that inhibit the differentiation of inflammatory myeloid cells affect antitumor immunity. Pharmacologic inhibition of Bruton's tyrosine kinase (BTK) and the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase (IDO) or deletion of Btk or Ido1 allowed robust differentiation of inflammatory Ly6c+CD103+ DCs during chemotherapy, promoting antitumor T cell responses and inhibiting tumor growth. Immature Ly6c+c-kit+ precursor cells had epigenetic profiles similar to conventional DC precursors; deletion of Btk or Ido1 promoted differentiation of these cells. Mechanistically, a BTK-IDO axis inhibited a tryptophan-sensitive differentiation pathway driven by GATOR2 and mTORC1, and disruption of the GATOR2 in monocyte-lineage precursors prevented differentiation into inflammatory DCs in vivo. IDO-expressing DCs and monocytic cells were present across a range of human tumors. Thus, a BTK-IDO axis represses differentiation of inflammatory DCs during chemotherapy, with implications for targeted therapies.

Light Chain Multiple Myeloma: High Serum Free Light Chain Concentrations Portend Renal Damage and Poorer Survival.

BACKGROUND: Monoclonal immunoglobulins provide an indication of the tumor burden in patients with plasma cell neoplasms. Higher concentrations of serum free light chains in light chain predominant multiple myeloma have been shown to correlate with a poorer outcome. We examined the correlations of serum free light chain concentrations in light chain myelomas with survival, estimated glomerular filtration rate (eGFR) and other clinical and pathological parameters. METHODS: Records of patients with light chain multiple myelomas were reviewed. Highest concentration of serum free light chains for each patient were plotted to ascertain an inflection/change point. Survival, eGFR, and other clinical and pathological parameters were compared between the low and high light chain concentration groups. RESULTS: Plotting serum free light chain concentrations revealed an inflection point at a concentration of 455 mg/L apportioning patients in to 2 subgroups: 39 patients with low light chain concentrations and 26 patients with high concentrations. The high concentration group had more unfavorable pathology in bone marrow examination in terms of higher neoplastic plasma cell burden and high-risk cytogenetics. The survival rate and eGFR in the high concentration group were significantly worse than in the low concentration group. CONCLUSIONS: As noted for light chain predominant multiple myeloma, high serum free light chain concentration in light chain multiple myelomas are associated with higher renal disease burden and shorter survival. Monitoring of serum free light chain concentrations and customizing treatments to address this parameter are warranted.

Immune subtraction for improved resolution in serum protein immunofixation electrophoresis and antibody isotype determination in a patient with autoantibody.

Heavy chain isotypes of low level monoclonal immunoglobulins are sometimes obscured in serum immunofixation electrophoresis (SIFE) by a heavy background of polyclonal immunoglobulins. However, accurate determination of the heavy chain isotype is essential for a complete diagnosis, as isotype determination of autoantibodies may have relevance in determining therapeutic procedures. Immune subtraction (IS) was employed in a patient with neuropathy and GD1a autoantibody. IS allowed identification of the cognate heavy chain related to a lambda light chain restriction noted on initial SIFE as well as isotype determination of the autoantibody. Antisera specific to individual heavy and light chains were used for depletion of specific immunoglobulin types. Depletion of kappa light chain associated immunoglobulins allowed unequivocal determination of the isotype of lambda light chain-associated low level monoclonal band to be IgG Lambda. Selective depletion of kappa, lambda, gamma and mu heavy chain immunoglobulins was employed to determine IgG Kappa isotype of the auto-antibody.

Engaging Pathology Residents in Clinical Chemistry: The Essential Ingredient Is a Committed Teacher.

BACKGROUND: Pathology residents are thought to show a lack of interest in clinical chemistry, therefore potentially graduating from training programs unprepared to function as laboratory directors and clinical consultants. METHODS: A structured program of tutorials based primarily on Henry's textbook, supplemented by recent review articles; a question bank of about 600 questions to emphasize key concepts; requirement for performing and presenting quality improvement projects; participation in on-site CAP inspections; review of reference laboratory test requests; and involving residents in scholarly activity have resulted in sustained, transferable, and significant improvements in engagement, knowledge, competence, and examination scores. RESULTS: The primary parameter for measuring change in resident competence and engagement were improvements in resident in-service examination (RISE) scores, publications in peer-reviewed journals, and receipt of awards. The revised program produced significant improvement in RISE scores in clinical chemistry, over and above the improvements in the general residency program. The residents were authors on 12 publications in peer-reviewed PubMed listed journals in the 5-year period since revision in the clinical chemistry curriculum compared to no publications in clinical chemistry in the 5-year period before the new curriculum. Over the past 2 years, 6 of the 11 publications by graduating residents were in clinical chemistry, and 6 of 7 awards for research were garnered by residents engaged in clinical chemistry investigations. All of the residents passed their clinical pathology boards on first attempt since the change compared to 2 failures in the prior 5-year period. CONCLUSIONS: The structured program described here is important as a template that could be adopted by any pathology training program. The question bank developed by this program is a valuable and transferable aid. However, success of such a program is dependent on the commitment of a knowledgeable, dedicated, and passionate teacher.

Streamlined Subclass-Specific Absolute Quantification of Serum IgG Glycopeptides Using Synthetic Isotope-Labeled Standards.

Absolute glycoproteomics quantification has drawn tremendous attention owing to its prospects in biomarker discovery and clinical implementation but is impeded by a general lack of suitable heavy isotope-labeled glycopeptide standards. In this study, we devised a facile chemoenzymatic strategy to synthesize a total of 36 human IgG glycopeptides attached with well-defined glycoforms, including 15 isotope-labeled ones with a mass increment of 6 Da to their native counterparts. Spiking of these standards into human sera enabled simplified, robust, and precise absolute quantification of IgG glycopeptides in a subclass-specific fashion. Additionally, the implementation of the absolute quantification approach revealed subclass-dependent alteration of serum IgG galactosylation and sialylation in colon cancer samples.

CD73 on cancer-associated fibroblasts enhanced by the A2B-mediated feedforward circuit enforces an immune checkpoint.

CD73, an ecto-5'-nucleotidase (NT5E), serves as an immune checkpoint by generating adenosine (ADO), which suppresses immune activation through the A2A receptor. Elevated CD73 levels in tumor tissues correlate with poor clinical outcomes. However, the crucial source of CD73 activity within the tumor microenvironment remains unspecified. Here, we demonstrate that cancer-associated fibroblasts (CAFs) constitute the prominent CD73hi population in human colorectal cancers (CRCs) and two CD73- murine tumor models, including a modified CRC. Clinically, high CAF abundancy in CRC tissues correlates strongly with elevated CD73 activity and poor prognosis. Mechanistically, CAF-CD73 expression is enhanced via an ADO-A2B receptor-mediated feedforward circuit triggered by tumor cell death, which enforces the CD73-checkpoint. Simultaneous inhibition of A2A and A2B pathways with CD73-neutralization synergistically enhances antitumor immunity in CAF-rich tumors. Therefore, the strategic and effective targeting of both the A2B-mediated ADO-CAF-CD73 feedforward circuit and A2A-mediated immune suppression is crucial for improving therapeutic outcomes.

Immune-mediated adverse events following influenza vaccine in cancer patients receiving immune checkpoint inhibitors.

OBJECTIVES: The emergence of immune checkpoint inhibitors has transformed treatment paradigms for various malignancies. Patients with cancer are at increased risk of complications and hospitalizations from influenza; therefore, it is recommended that they receive inactivated influenza vaccination. However, efficacy and safety of inactivated influenza vaccination in patients receiving immune checkpoint inhibitors is uncertain. The objective of this prospective case series was to evaluate the incidence of immune-mediated adverse events (imAEs) following inactivated influenza vaccination in patients receiving immune checkpoint inhibitors. Changes in cytokine and chemokine levels were also evaluated. METHODS: Patients receiving immune checkpoint inhibitors during the 2017-2018 influenza season were eligible for study participation. Peripheral blood samples were collected prior to administration of inactivated influenza vaccine and two post-vaccination time points. Evaluation of new or worsening imAEs occurred via patient questionnaire and review of medical records for 60 days following inactivated influenza vaccination. Baseline imAEs were evaluated from review of medical records for 60 days prior to inactivated influenza vaccination. Serum cytokines and chemokines were measured using a multiplex Luminex assay. RESULTS: Twenty-four patients were enrolled in this study. Seven patients experienced any grade imAE (one patient having 2) within 60 days following inactivated influenza vaccination. The majority were Grades 1-2, including rash (n = 3), hypothyroidism, myalgia, and colitis (n = 1 each). Two patients experienced severe imAEs (grade 3 nephritis and grade 4 diabetes). No significant changes (p > 0.05) in serum cytokine or chemokine concentrations were observed. CONCLUSIONS: Although small, our study suggests that inactivated influenza vaccine may be safely administered to patients receiving immune checkpoint inhibitors. The majority of imAEs following inactivated influenza vaccination were Grades 1-2 and did not require changes in immune checkpoint inhibitor therapy.

Clinical Parameters Outperform Molecular Subtypes for Predicting Outcome in Bladder Cancer: Results from Multiple Cohorts, Including TCGA.

PURPOSE: Studies indicate that molecular subtypes in muscle invasive bladder cancer predict the clinical outcome. We evaluated whether subtyping by a simplified method and established classifications could predict the clinical outcome. MATERIALS AND METHODS: We subtyped institutional cohort 1 of 52 patients, including 39 with muscle invasive bladder cancer, an Oncomine™ data set of 151 with muscle invasive bladder cancer and TCGA (The Cancer Genome Atlas) data set of 402 with muscle invasive bladder cancer. Subtyping was done using simplified panels (MCG-1 and MCG-Ext) which included only transcripts common in published studies and were analyzed for predicting metastasis, and cancer specific, overall and recurrence-free survival. TCGA data set was further analyzed using the Lund taxonomy, the Bladder Cancer Molecular Taxonomy Group Consensus and TCGA 2017 mRNA subtype classifications. RESULTS: Muscle invasive bladder cancer specimens from cohort 1 and the Oncomine data set showed intratumor heterogeneity for transcript and protein expression. MCG-1 subtypes did not predict the outcome on univariate or Kaplan-Meier analysis. On multivariate analysis N stage (p ≤0.007), T stage (p ≤0.04), M stage (p=0.007) and/or patient age (p=0.01) predicted metastasis, cancer specific and overall survival, and/or the cisplatin based adjuvant chemotherapy response. In TCGA data set publications showed that subtypes risk stratified patients for overall survival. Consistently the MCG-1 and MCG-Ext subtypes were associated with overall but not recurrence-free survival on univariate and Kaplan-Meier analyses. TCGA data set included 21 low grade specimens of the total of 402 and subtypes associated with tumor grade (p=0.005). However, less than 1% of muscle invasive bladder cancer cases are low grade. In only high grade specimens the MCG-1 and MCG-Ext subtypes could not predict overall survival. On univariate analysis subtypes according to the Bladder Cancer Molecular Taxonomy Group Consensus, TCGA 2017 and the Lund taxonomy were associated with tumor grade (p <0.0001) and overall survival (p=0.01 to <0.0001). Regardless of classification, subtypes had about 50% to 60% sensitivity and specificity to predict overall and recurrence-free survival. On multivariate analyses N stage and lymphovascular invasion consistently predicted recurrence-free and overall survival (p=0.039 and 0.003, respectively). CONCLUSIONS: Molecular subtypes reflect bladder tumor heterogeneity and are associated with tumor grade. In multiple cohorts and subtyping classifications the clinical parameters outperformed subtypes for predicting the outcome.

A chemokine/chemokine receptor signature potentially predicts clinical outcome in colorectal cancer patients.

BACKGROUND: Differential expression of chemokines/chemokine receptors in colorectal cancer (CRC) may enable molecular characterization of patients' tumors for predicting clinical outcome. OBJECTIVE: To evaluate the prognostic ability of these molecules in a CRC cohort and the CRC TCGA-dataset. METHODS: Chemokine (CXCL-12α, CXCL-12ß, IL-17A, CXCL-8, GM-CSF) and chemokine receptor (CXCR-4, CXCR-7) transcripts were analyzed by RT-qPCR in 76 CRC specimens (normal: 27, tumor: 49; clinical cohort). RNA-Seq data was analyzed from the TCGA-dataset (n= 375). Transcript levels were correlated with outcome; analyses: univariate, multivariable, Kaplan-Meier. RESULTS: In the clinical cohort, chemokine/chemokine receptor levels were elevated 3-10-fold in CRC specimens (P⩽ 0.004) and were higher in patients who developed metastasis (P= 0.03 - < 0.0001). CXCR-4, CXCR-7, CXCL-12α, CXCL-8, IL-17 and GM-CSF levels predicted metastasis (P⩽ 0.0421) and/or overall survival (OS; P⩽ 0.0373). The CXCR-4+CXCR-7+CXCL-12 marker (CXCR-4/7+CXCL-12 (α/b) signature) stratified patients into risk for metastasis (P= 0.0014; OR, 2.72) and OS (P= 0.0442; OR, 2.7); sensitivity: 86.67%, specificity: 97.06%. In the TCGA-dataset, the CXCR-4/7+CXCL-12 signature predicted metastasis (P= 0.011; OR, 2.72) and OS (P= 0.0006; OR: 4.04). In both datasets, the signature was an independent predictor of clinical outcome. CONCLUSIONS: Results of 451 specimens from both cohorts reveal that the CXCR-4/7+CXCL-12 signature potentially predicts outcome in CRC patients and may allow earlier intervention.

Multi-omics Integration Analysis Robustly Predicts High-Grade Patient Survival and Identifies CPT1B Effect on Fatty Acid Metabolism in Bladder Cancer.

PURPOSE: The perturbation of metabolic pathways in high-grade bladder cancer has not been investigated. We aimed to identify a metabolic signature in high-grade bladder cancer by integrating unbiased metabolomics, lipidomics, and transcriptomics to predict patient survival and to discover novel therapeutic targets. EXPERIMENTAL DESIGN: We performed high-resolution liquid chromatography mass spectrometry (LC-MS) and bioinformatic analysis to determine the global metabolome and lipidome in high-grade bladder cancer. We further investigated the effects of impaired metabolic pathways using in vitro and in vivo models. RESULTS: We identified 519 differential metabolites and 19 lipids that were differentially expressed between low-grade and high-grade bladder cancer using the NIST MS metabolomics compendium and lipidblast MS/MS libraries, respectively. Pathway analysis revealed a unique set of biochemical pathways that are highly deregulated in high-grade bladder cancer. Integromics analysis identified a molecular gene signature associated with poor patient survival in bladder cancer. Low expression of CPT1B in high-grade tumors was associated with low FAO and low acyl carnitine levels in high-grade bladder cancer, which were confirmed using tissue microarrays. Ectopic expression of the CPT1B in high-grade bladder cancer cells led to reduced EMT in in vitro, and reduced cell proliferation, EMT, and metastasis in vivo. CONCLUSIONS: Our study demonstrates a novel approach for the integration of metabolomics, lipidomics, and transcriptomics data, and identifies a common gene signature associated with poor survival in patients with bladder cancer. Our data also suggest that impairment of FAO due to downregulation of CPT1B plays an important role in the progression toward high-grade bladder cancer and provide potential targets for therapeutic intervention.

Primary tumor-induced immunity eradicates disseminated tumor cells in syngeneic mouse model.

Although clinically apparent metastasis is associated with late stages of cancer development, micro-metastatic dissemination may be an early event. However, the fate of these early disseminated tumor cells (DTC) remains elusive. We show that despite their capacity to disseminate into secondary organs, 4T1 tumor models develop overt metastasis while EMT6-tumor bearing mice clear DTCs shed from primary tumors as well as those introduced by intravenous (IV) injection. Following the surgical resection of primary EMT6 tumors, mice do not develop detectable metastasis and reject IV-injected tumor cells. In contrast, these cells readily grow and metastasize in immuno-deficient athymic or Rag2-/- mice, an effect mimicked by CD8+ T-cell depletion in immunocompetent mice. Furthermore, recombinant G-CSF or adoptive transfer of granulocytic-MDSCs isolated from 4T1 tumor-bearing mice, induce metastasis by suppressing CD8+ T-cells in EMT6-primed mice. Our studies support the concept of immune surveillance providing molecular insights into the immune mechanisms during tumor progression.