Energy metabolism and cell motility defect in NK-cells from patients with hepatocellular carcinoma.

Functional rescue of NK-cells in solid tumors represents a central aim for new immunotherapeutic strategies. We have conducted a genomic, phenotypic and functional analysis of circulating NK-cells from patients with HCV-related liver cirrhosis and hepatocellular carcinoma. NK-cells were sorted from patients with HCC or liver cirrhosis and from healthy donors. Comparative mRNA gene expression profiling by whole-human-genome microarrays of sorted NK-cells was followed by phenotypic and functional characterization. To further identify possible mediators of NK-cell dysfunction, an in vitro model using media conditioned with patients' and controls' plasma was set up. Metabolic and cell motility defects were identified at the genomic level. Dysregulated gene expression profile has been translated into reduced cytokine production and degranulation despite a prevalent phenotype of terminally differentiated NK-cells. NKG2D-downregulation, high SMAD2 phosphorylation and other phenotypic and molecular alterations suggested TGF-ß as possible mediator of this dysfunction. Blocking TGF-ß could partially restore functional defects of NK-cells from healthy donors, exposed to TGF-ß rich HCC patients' plasma, suggesting that TGF-ß among other molecules may represent a suitable target for immunotherapeutic intervention aimed at NK-cell functional restoration. By an unbiased approach, we have identified energy metabolism and cell motility defects of circulating NK-cells as main mechanisms responsible for functional NK-cell impairment in patients with hepatocellular carcinoma. This opens the way to test different approaches to restore NK-cell response in these patients.

Counter-regulation of regulatory T cells by autoreactive CD8+ T cells in rheumatoid arthritis.

The mechanisms whereby autoreactive T cells escape peripheral tolerance establishing thus autoimmune diseases in humans remain an unresolved question. Here, we demonstrate that autoreactive polyfunctional CD8+ T cells recognizing self-antigens (i.e., vimentin, actin cytoplasmic 1, or non-muscle myosin heavy chain 9 epitopes) with high avidity, counter-regulate Tregs by killing them, in a consistent percentage of rheumatoid arthritis (RA) patients. Indeed, these CD8+ T cells express a phenotype and gene profile of effector (eff) cells and, upon antigen-specific activation, kill Tregs indirectly in an NKG2D-dependent bystander fashion in vitro. This data provides a mechanistic basis for the finding showing that AE-specific (CD107a+) CD8+ T killer cells correlate, directly with the disease activity score, and inversely with the percentage of activated Tregs, in both steady state and follow-up studies in vivo. In addition, multiplex immunofluorescence imaging analyses of inflamed synovial tissues in vivo show that a remarkable number of CD8+ T cells express granzyme-B and selectively contact FOXP3+ Tregs, some of which are in an apoptotic state, validating hence the possibility that CD8+ Teff cells can counteract neighboring Tregs within inflamed tissues, by killing them. Alternatively, the disease activity score of a different subset of patients is correlated with the expansion of a peculiar subpopulation of autoreactive low avidity, partially-activated (pa)CD8+ T cells that, despite they conserve the conventional naïve (N) phenotype, produce high levels of tumor necrosis factor (TNF)-α and exhibit a gene expression signature of a progressive activation state. Tregs directly correlate with the expansion of this autoreactive (low avidity) paCD8+ TN cell subset in vivo, and efficiently control their differentiation rather their proliferation in vitro. Interestingly, autoreactive high avidity CD8+ Teff cells or low avidity paCD8+ TN cells are significantly expanded in RA patients who would become non-responders or patients who would become responders to TNF-α inhibitor therapy, respectively. These data provide evidence of a previously undescribed role of such mechanisms in the progression and therapy of RA.

Immune landscape of hepatocellular carcinoma microenvironment: Implications for prognosis and therapeutic applications.

The development of immunotherapy for solid tumours has boosted interest in the contexture of tumour immune microenvironment (TIME). Several lines of evidence indicate that the interplay between tumour cells and TIME components is a key factor for the evolution of hepatocellular carcinoma (HCC) and for the likelihood of response to immunotherapeutics. The availability of high-resolution methods will be instrumental for a better definition of the complexity and diversity of TIME with the aim of predicting disease outcome, treatment response and possibly new therapeutic targets. Here, we review current knowledge about the immunological mechanisms involved in shaping the clinical course of HCC. Effector cells, regulatory cells and soluble mediators are discussed for their role defining TIME and as targets for immune modulation, together with possible immune signatures for optimization of immunotherapeutic strategies.

Carbohydrate-deficient transferrin: utility of HPLC in handling atypical samples uninterpretable by capillary electrophoresis.

AIMS: Carbohydrate-deficient transferrin (CDT) is a marker of chronic alcohol abuse. Uninterpretable (atypical) CDT patterns have been detected by both capillary electrophoresis (CE) and HPLC. The aim of this study was to evaluate the performance of HPLC as a second-line test for the interpretation of most frequent atypical CDT profiles detected by CE. METHODS: CDT was analyzed by CE (Capillarys 2, Sebia) on 9120 consecutive samples in a routine laboratory setting during a 2-year period. A commercial method (ClinRep CDT kit, Recipe) was employed to retest 123 (1.4%) samples with atypical CDT patterns on a Prominence LC-20AT HPLC (Shimadzu). RESULTS: CE-uninterpretable samples were categorized as having low transferrin (Tf) concentration (LT; n = 42, 0.5%), di-trisialotransferrin bridging (D-TB; n = 63, 0.7%) or atypical peak profile (APP; n = 18, 0.2%). CDT was detectable by HPLC in 58 of 123 (47%) samples including 21of 42 (50%) with LT, 27 of 63 (43%) with D-TB and 10 of 18 (56%) with APP. CONCLUSIONS: Second-line HPLC testing reduced uninterpretable samples by 47%, with similar rates of improvement regardless of the type of CDT pattern. The usefulness of HPLC as a second-line test for CDT should be evaluated according to cost-benefit considerations in the context of each laboratory.

Optimisation of triple therapy for patients with chronic hepatitis C: a systematic review.

BACKGROUND: Triple therapy with Pegylated-Interferon α (PEG-IFNα)/Ribavirin (RBV) and Boceprevir (Boc) or Telaprevir (Tel) significantly improved sustained virological response (SVR) rates for patients with genotype 1 HCV infection compared to PEG-IFNα/RBV alone (dual therapy). However, less is known about factors associated with rates of SVR and of adverse events (AEs). MATERIAL AND METHODS: The aim of this systematic review was to evaluate the evidence regarding the factors affecting response and rate of AEs associated with triple therapy. We performed systematic electronic searches in Medline, Embase, Scopus and Central as well as a list of reference literature. We included randomised controlled trials examining triple therapy compared with dual therapy and reporting data according to patients features and about AEs. Odds ratios (OR) were pooled using either fixed or random effect model, as appropriate. RESULTS: We included data from 14 studies. Treatment with triple therapy increased SVR rate compared to dual therapy especially in patients previously treated with PEG-IFNα/RBV and with increased pretreatment alanine aminotransferase (ALT) levels. Higher rate of serious AEs and treatment discontinuation due to AEs was also observed particularly in treatment-experienced patients. CONCLUSIONS: The present study shows how improved results of triple therapy are mainly observed in some patients' subsets and are accompanied by increased risk of AEs compared to dual therapy. These results might be useful for optimising treatment of chronic hepatitis C when IFN-free regimens are unavailable.

Carbohydrate-Deficient Transferrin Determination in a Clinical Setting: Consistency Between Capillary Electrophoresis Assays and Utility of HPLC as a Confirmatory Test.

BACKGROUND: Carbohydrate-deficient transferrin (CDT) is used to assess chronic alcohol consumption in administrative and forensic context. The aim of the present study was the optimization of the diagnostic strategy for CDT determination in a clinical laboratory setting. METHODS: Two capillary zone electrophoresis (CZE) assays, the CEofix CDT (Analis, Suarlée, Belgium) run on single capillary MDQ instrument and the muticapillary (Sebia, Lisses, France), were compared as screening methods and a commercial high-performance liquid chromatography (HPLC) assay (Recipe, Munich, Germany) was used for confirmation. RESULTS: In total, 367 serum samples were analyzed by both CZE assays with concordant classification in 92% of cases. All discordant samples were classified as negative by HPLC, as did 2/3 of those that could not be classified by either CZE assay. Classification of samples with CDT values close to cut-off by CZE was confirmed by HPLC in 95-100% of negative samples but only in 28.6-33.3% of positive samples. CONCLUSIONS: Both CZE assays proved suitable for CDT screening. HPLC was useful for discriminating CDT value in most of samples that could not be interpreted by CZE due to analytical interferences. Considering the implication of CDT testing, HPLC assay may also be helpful for the confirmation of positive results close to the cut-off value of CZE assays.

High CD49a+ NK cell infiltrate is associated with poor clinical outcomes in Hepatocellular Carcinoma.

NK cells infiltrating Hepatocellular Carcinoma (HCC) may express residency markers such as Integrin Subunit Alpha 1 (CD49a) that have been associated with nurturing functions in the decidua, and characterized by the production of angiogenic factors as well as loss of cytotoxicity. CIBERSORT, a computational analysis method for quantifying cell fractions from bulk tissue gene expression profiles, was used to estimate the infiltrating immune cell composition of the tumor microenvironment from gene expression profiles of a large cohort of 225 HCCs in the public GEO database. Decidual-like CD49a+ NK cells, in addition to another 22 immune cell populations, were characterized and thoroughly investigated so that HCC cell heterogeneity in a large cohort of 225 HCCs from the public GEO database could be studied. An inverse correlation of the expression of CD49a+ NK-cells and CD8+ T-cells suggested a negative association with clinical outcomes. This result was confirmed in a further validation cohort of 100 HCC patients from The Cancer Genome Atlas, Liver Hepatocellular Carcinoma (TCGA-LIHC). Cox regression analysis did not identify CD49a+ cells as a variable independently associated with survival. However, a more abundant infiltrate of this subset was present in patients at a more advanced pathological and clinical HCC stage. In conclusion, we found that NK cells, with a decidual-like gene expression profile, are enriched in HCC, and their abundance increases not only in tumor size but also at advanced stages of the disease suggesting that these cells play a role in tumor growth. For this reason, these NK cells may represent a possible new target for immunotherapeutic approaches in HCC.

A system consisting of cation-exchange chromatography, combined surface-activated chemical and electrospray ionization, and ion-trap analysis for the analysis of 8-oxo-7,8-dihydro-2'-deoxyguanosine in urine.

RATIONALE: 8-Hydroxy-2'-deoxyguanosine has served as a biomarker for oxidative damage to DNA from different types of biological samples, and various techniques have been used to analyze it. In particular, liquid chromatography coupled to mass spectrometry has been used to identify 8-hydroxy-2'-deoxyguanosine in urine samples. Usually, a triple quadrupole analyzer and multiple reaction monitoring have been employed for its detection. Only a few studies have used a less expensive ion-trap analyzer instead. METHODS: We have developed a new liquid chromatography/mass spectrometry procedure that incorporates cation-exchange chromatography in conjunction with surface-activated and electrospray ionization with an ion trap analyzer for the mass spectral step. RESULTS: The combination of two ionization sources reduced the matrix effect arising from in-source reactions, thus increasing the sensitivity to levels comparable with those obtained by triple quadrupole analyzers. CONCLUSIONS: This new method for 8-hydroxy-2'-deoxyguanosine detection provided increased sensitivity and reduced chemical noise, using a less expensive, stable and accurate mass spectrometric technology.

Targeting Stress Sensor Kinases in Hepatocellular Carcinoma-Infiltrating Human NK Cells as a Novel Immunotherapeutic Strategy for Liver Cancer.

Natural killer (NK) cells may become functionally exhausted entering hepatocellular carcinoma (HCC), and this has been associated with tumor progression and poor clinical outcome. Hypoxia, low nutrients, immunosuppressive cells, and soluble mediators characterize the intratumor microenvironment responsible for the metabolic deregulation of infiltrating immune cells such as NK cells. HCC-infiltrating NK cells from patients undergoing liver resection for HCC were sorted, and genome-wide transcriptome profiling was performed. We have identified a marked general upregulation of gene expression profile along with metabolic impairment of glycolysis, OXPHOS, and autophagy as well as functional defects of NK cells. Targeting p38 kinase, a stress-responsive mitogen-activated protein kinase, we could positively modify the metabolic profile of NK cells with functional restoration in terms of TNF-α production and cytotoxicity. We found a metabolic and functional derangement of HCC-infiltrating NK cells that is part of the immune defects associated with tumor progression and recurrence. NK cell exhaustion due to the hostile tumor microenvironment may be restored with p38 inhibitors with a selective mechanism that is specific for tumor-infiltrating-not affecting liver-infiltrating-NK cells. These results may represent the basis for the development of a new immunotherapeutic strategy to integrate and improve the available treatments for HCC.

Translating pharmacogenetics into clinical practice: interleukin (IL)28B and inosine triphosphatase (ITPA) polymophisms in hepatitis C virus (HCV) infection.

Hepatitis C virus (HCV) infection is frequently characterized by evolution to chronicity and by a variable clinical course of the disease. The clinical heterogeneities of HCV infection and the imperfect predictability of the response to interferon (IFN) have suggested the need to search for a genetic basis of clinical features. This led to the discovery of genetic polymorphisms playing a major role in the evolution of infection, as well as on treatment response and adverse effects. This review will cover recent reports on the subject, focusing on the potential use of the new genetic markers in the diagnostic algorithm for the stratification of patients for personalized antiviral regimens.

Intratumor Regulatory Noncytotoxic NK Cells in Patients with Hepatocellular Carcinoma.

Previous studies support the role of natural killer (NK) cells in controlling hepatocellular carcinoma (HCC) progression. However, ambiguity remains about the multiplicity and the role of different NK cell subsets, as a pro-oncogenic function has been suggested. We performed phenotypic and functional characterization of NK cells infiltrating HCC, with the corresponding nontumorous tissue and liver from patients undergoing liver resection for colorectal liver metastasis used as controls. We identified a reduced number of NK cells in tumors with higher frequency of CD56BRIGHTCD16- NK cells associated with higher expression of NKG2A, NKp44, and NKp30 and downregulation of NKG2D. Liver-resident (CXCR6+) NK cells were reduced in the tumors where T-bethiEomeslo expression was predominant. HCCs showed higher expression of CD49a with particular enrichment in CD49a+Eomes+ NK cells, a subset typically represented in the decidua and playing a proangiogenic function. Functional analysis showed reduced TNF-α production along with impaired cytotoxic capacity that was inversely related to CXCR6-, T-bethiEomeslo, and CD49a+Eomes+ NK cells. In conclusion, we identified a subset of NK cells infiltrating HCC, including non-liver-resident cells that coexpressed CD49a and Eomes and showed reduced cytotoxic potential. This NK cell subset likely plays a regulatory role in proangiogenic function.

[Analytical quality in biological monitoring of workers exposed to chemicals: experience of the Prevention and Safety at the Workplace Service in Modena]. / Qualità del dato analitico nel monitoraggio biologico dei lavoratori esposti a sostanze chimiche: esperienza del Servizio Prevenzione e Sicurezza Ambienti di Lavoro della Azienda USL di Modena.

BACKGROUND: The quality of laboratory data is one of the main factors in guaranteeing efficacy of biological monitoring. OBJECTIVES: To analyze the quality of laboratory data used for biological monitoring of exposed workers. METHODS: A survey involving 18 companies employing 945 workers in the area of Modena, Italy, was carried out in 2008. RESULTS: Most of the 9 private laboratories receiving biological samples did not perform directly part or all of the laboratory assessments requested, but this was not indicated in the final report. Major problems were observed in the application of internal quality control, and only one laboratory participated in external quality assessment for blood lead measurements. CONCLUSIONS: Our results raise major concerns on the traceability and reliability of laboratory assessments performed for biomonitoring of exposed workers. Systematic evaluation of the quality of analytical data would be highly recommendable.

New Direct-Acting Antivirals for the Treatment of Patients With Hepatitis C Virus Infection: A Systematic Review of Randomized Controlled Trials.

BACKGROUND: New direct-acting antiviral agents (DAAs) approved for the treatment of patients infected by Hepatitis C virus (HCV) are well tolerated and increase sustained virological response (SVR) rate. We summarize current evidence on the efficacy and safety from comparative randomized controlled trials (RCTs) of DAAs. METHODS: We systematically searched MEDLINE, Embase, Scopus, CENTRAL, and Lilacs as well as a list of reference literature. We included RCTs comparing DAAs with placebo or active control and reporting response rates and adverse events according to antiviral regimens. Risk ratios (RRs) were pooled as appropriate. We assessed the risk of bias of included studies and graded the quality of evidence according to the GRADE method. RESULTS: We included 28 RCTs, enrolling more than 7000 patients. The quality of evidence was generally low. Twelve-week treatment with DAAs in naïve patients significantly increased SVR12 and SVR24 compared with placebo (RR 1.4, 95% CI 1.3-1.6; RR 1.5, 95% CI 1.4-1.6, respectively). This means that for every 1000 patients, 240 or 260 more patients experienced SVR12 or SVR24 if treated with any DAAs. We could not find RCTs assessing progression of liver disease or development of hepatocellular carcinoma. DAAs were not associated with higher incidence of serious adverse events or discontinuation due to adverse events. CONCLUSIONS: This systematic review confirms that new DAAs are more effective in inducing SVR than placebo. Outside clinical trials, in real word, HCV cure with DAA regimens occurs in less than 90% of patients, so further comparative evaluations are needed to establish their long-term effects.

Immune Gene Expression Profile in Hepatocellular Carcinoma and Surrounding Tissue Predicts Time to Tumor Recurrence.

BACKGROUND: The antitumor immune response may play a major role in the clinical outcome of hepatocellular carcinoma (HCC). We characterized the liver immune microenvironment by direct hybridization of RNA extracted from HCC and nontumorous tissues. METHODS: RNA was extracted from frozen liver tissue samples of HCC (T; n = 30) and nontumorous tissues (NT; n = 33) obtained from 38 patients. Matched samples were available for 25 patients. The immune gene expression profile was analyzed with the nCounter GX Human Immunology v2 system (NanoString Technologies), which detects the expression levels of 579 immune response-related genes simultaneously. RESULTS: Since the immune gene expression profile of T and NT tissues was significantly different, the prognostic relevance of the liver immune microenvironment was evaluated in the T and NT samples separately. Unsupervised clustering detected two main clusters of immune gene expression both in T and in NT liver samples. In both cases, the expression clusters identified groups of patients with a significantly different median time to HCC recurrence (TTR) but similar overall survival. Based on T tissue, two groups with median TTR of 19 and 127 months, respectively, were detected (p < 0.005). Expression of genes related to T-cell activation was associated with longer TTR. The analysis of NT tissue discriminated subsets of patients with median TTR of 22 and 68 months (p < 0.05). In contrast to T tissue, a predominant inflammatory immune environment was associated with shorter TTR. CONCLUSIONS: Immune gene expression profiles predictive of TTR could be identified both in HCC and in adjacent cirrhotic tissues. Longer TTR was associated with overexpression in T tissue and downregulation in NT tissue of the immune response and of inflammation-related genes.

Comparison of commercial and in-house Real-time PCR assays for quantification of Epstein-Barr virus (EBV) DNA in plasma.

BACKGROUND: Epstein-Barr virus (EBV) DNA load monitoring is known to be useful for the diagnosis and monitoring of EBV-associated diseases. The aim of this study is to compare the performance of two real-time PCR assays for EBV DNA: a commercial kit as the Q-EBV Real-Time System (Q-EBV PCR, Amplimedical, Turin, Italy) and an in-house assay (EBV RQ-PCR). RESULTS: The range of linearity and the degree of precision of the two assays were similar. The clinical sensitivity of Q-EBV PCR was higher for reference samples containing less than 1,000 EBV DNA copies/ml. The absolute quantitative results of the two methods were statistically correlated (R2 = 0.7789; p < 0.0001), with the systematic overestimation by EBV RQ-PCR possibly linked to different amplification efficiency in calibration standards. CONCLUSION: Both the commercial and the in-house assay may be appropriate for clinical use, but common standards are advisable for comparable absolute values, as these would improve the clinical utility of EBV DNA load measurement.

Relationship between pp65 antigenemia levels and real-time quantitative DNA PCR for Human Cytomegalovirus (HCMV) management in immunocompromised patients.

BACKGROUND: Quantitative real-time PCR assays, which are more rapid and practical than pp65 antigenemia determination, are progressively becoming the preferred method for monitoring Human Cytomegalovirus (HCMV) reactivation. However, the relationship between HCMV DNA and antigenemia levels is still under investigation. The aim of this study was to analyse the relationship between HCMV DNA and pp65 antigenemia levels in order to identify clinically useful threshold values for the management of patients. METHODS: 475 consecutive samples from 156 immunosuppressed patients were tested for HCMV by pp65 antigenemia and Real-time PCR assay. RESULTS: 136 out of 475 consecutive samples derived from 48 patients showed evidence of HCMV infection. HCMV DNA was detected in 106 samples, pp65 antigen in 3, and both markers in 27. pp65 antigen detection was associated with higher HCMV DNA levels. The cut-off HCMV DNA level that best predicted pp65 antigenemia in this series of samples was 11,500 copies/ml, but different threshold levels could be observed for specific groups of patients. HCMV disease was observed in 5 out of 48 patients with active HCMV infection. The presence of clinical symptoms was associated with positive pp65 and with higher antigenemia levels. Higher HCMV DNA load at the onset of viral replication was correlated to the development of clinical symptoms. CONCLUSION: Both pp65 antigenemia and HCMV DNA load can be useful for the prospective monitoring of immunocompromised subjects. Specific cut-off levels capable of triggering preemptive antiviral treatment should be determined in accordance to the type of test used and the characteristics of patients and prospectively validated.

Natural killer cells phenotypic characterization as an outcome predictor of HCV-linked HCC after curative treatments.

NK-cell number and function have been associated with cancer progression. A detailed analysis of phenotypic and functional characteristics of NK-cells in HCC is still lacking. NK-cell function is regulated by activating and inhibitory receptors determined by genetic factors and engagement with cognate ligands on transformed or infected cells. We evaluated phenotypic and functional characteristic of NK-cells in HCC patients undergoing curative treatment in relation to clinical outcome. NK-cells from 70 HCC patients undergoing resection or ablative treatment, 18 healthy volunteers and 12 cirrhotic patients with HCV-infection (controls) were phenotypically characterized. Unsupervised clustering based on the frequency of cells expressing different phenotypic NK-cell markers segregated HCC patients into different cohorts that were compared for outcome. NK-cell cytokine production and cytotoxicity were compared between cohorts with different overall survival (OS) and time to disease recurrence (TTR). By multivariate analysis, age, Child-Pugh class and NK-cell phenotypic clustering could independently identify patients with significantly different OS. NK-cells from patients with better outcome expressed higher levels of cytotoxic granules and CD3ζ and lower levels of natural cytotoxic receptors (NCRs) that were co-expressed with the inhibitory receptor NKG2A known to negatively regulate NCR function. Cytotoxic function and IFNγ production were significantly lower in the cohort of patients with worse outcome compared to controls (p < 0.05). Our results show a role for NK-cells in the control of HCC progression and survival providing the basis for the development of immunotherapeutic strategies to potentiate NK-cell response.

Interferon lambda-3 is not associated with clinical outcome in patients with HCV-induced compensated cirrhosis: a long-term cohort study.

BACKGROUND: Interferon Lambda-3 (IFN-λ3) gene polymorphism is associated with spontaneous clearance of hepatitis C virus (HCV) and response to IFN-based therapy (IFN). However, very few data are available about its value in predicting sustained virologic response (SVR) in patients with cirrhosis, and whether IFN-λ3 genotype influences liver disease progression remains unclear. METHODS: We determined IFN-λ3 genotype by PCR in a cohort of patients with compensated HCV-related cirrhosis, enrolled between 1989 and 1992. Person-years follow-up was calculated for each individual from the date of enrolment to the development of first episode of decompensation, HCC, liver transplant, death or end of follow-up. The follow-up of patients who achieved SVR was censored at the time of IFN initiation. Kaplan-Meier curves and Cox regression analyses were used to assess the association between IFN-λ3 genotype and clinical outcome. RESULTS: IFN-λ3 was determined in 264 patients (52% males, mean age 57±8 years, 67% HCV genotype (G)1, while CC, CT and TT genotypes were 36%, 50% and 14%, respectively. During a median follow-up of 14.8 years, 149 (56%) patients received IFN. Overall, SVR was achieved in 31 (21%) patients, 40% among those with CC genotype (22% in G1 and 61% in G2, respectively) compared to 10% and 13% among patients with CT and TT genotypes (p<0.0001). Univariate and multivariate analyses found no association between IFN-λ3 (CC vs. non-CC genotype) and disease progression. CONCLUSION: IFN-λ3 determination is fundamental for allocating cirrhotic patients to be treated with IFN, while it has no value in predicting the outcome of the disease.