Biomarkers and Surrogate Endpoints: Advancing Clinical Research and Applications

Severe Infections and Septic Shock

Objectives

• Review state of current biomarkers and surrogate endpoints in sepsis clinical research

- What is the experience to date in clinical trials with specific biomarkers and secondary endpoints?

- What is the association of biomarkers and surrogate endpoints with mortality or organ failure?

- How do physiologic endpoints (e.g., reversal of shock, development of organ failure) interact with biomarkers and mortality?

- Have these physiologic endpoints been affected by the experimental therapies?

· Discuss role of biomarkers and surrogate endpoints in enhancing development of trials of novel therapies for septic shock and review their role in preclinical and clinical studies in measuring therapeutic and adverse effects of agents

- Can substitution of biomarkers for clinical endpoints be used to evaluate the safety and efficacy of a novel therapy?

- Can information be obtained from existing databases of completed trials to validate biomarkers?

- Can biomarkers be applied as a surrogate endpoints in Phase III trials of therapies for sepsis?

· Discuss the role of biomarkers and surrogate endpoints in enhancing development of trials of novel therapies for septic shock and review their role in preclinical and clinical studies in measuring therapeutic and adverse effects of agents.

-What strategies are necessary to incorporate novel technologies (e.g., functional and structural imaging, patterns of gene expression via cDNA microarray technology, gene polymorphisms, cell signaling markers, circulating biomarkers, functional assays such as proinflammatory activity of body fluids, banked tissues from clinical trials) in the development of novel biomarkers?

- How should the new technologies be validated in preclinical and clinical applications?

- What resources are available to fund biomarker research, including new technology application, development of tissue and blood banks from clinical trials, and consortium development?

Agenda

Moderators: Thomas R. Martin, M.D., University of Washington and Seattle Veterans Affairs Medical Center

Cytokine Balance in Acute Respiratory Distress Syndrome: Implications for Detecting Acute Lung Injury

Prospects for Functional Immune Assessment in Severe Infections and Septic Shock

Validating Biomarkers and Ascertaining Their Relationship to Clinical Endpoints

Severity of Infectious Challenge Alters the Effects of Anti-Inflammatory Agents in Sepsis

Discussants: Jay Siegel, M.D., Center for Biologics Evaluation and Research, U.S. Food and Drug Administration

ABSTRACTS

Overview of Biomarkers and Secondary Endpoints in Sepsis

Major breakthroughs for the treatment of sepsis remain elusive. More than 10,000 patients (21 studies) have been enrolled in trials of nonsteroidal antiinflammatory agents in septic shock, and no agent has altered outcomes. When the treatment effects from these trials are pooled, a small (3 percent) beneficial effect, equal to a 7‑percent reduction in mortality, is found (Natanson et al. 1998). These data suggest that mediator‑specific anti‑inflammatory therapies as currently applied (e.g., patient selection, dose, duration of therapy) have at most only small beneficial effects. Power analysis suggests that demonstrating these modest effects would require large trials (more than 5,000 patients). To date, however, clinical parameters (e.g., severity of illness scores) or biologic markers (e.g., blood cytokine and endotoxin levels or cell antigen expression) have not proven useful for selecting patients for these inflammation‑ modifying therapies (Reinhart et al. 1996; Abraham et al. 1998). Although biomarkers in some diseases correlate with pathogenesis and outcome (e.g., CD4 cell counts in HIV infection), syndromes such as sepsis are more difficult to characterize because the clinical importance of biomarkers may depend on their context (e.g., tumor neurosis factor [TNF] has beneficial and harmful effects) and the net effects of other signaling molecules (Brandtzaeg et al. 1996). Identifying new host or microbial‑derived biomarkers, refining the use of existing markers, and developing models that reflect the complex nature of these interactions will be important in future clinical trial development.

Abraham E, Anzueto A, Gutierrez G, Tessler S, San Pedro G, Wunderink R, Dal Nogare A, Nasraway S, Berman S, Cooney R, Levy H, Baughman R, Rumbak M, Light RB, Poole L, Allred R, Constant J, Pennington J, Porter S. Double‑blind randomised controlled trial of monoclonal antibody to human tumor necrosis factor in treatment of septic shock. NORASEPT II Study Group. Lancet 1998;351:929‑933.

Brandtzaeg P, Osnes L, Ovstebo R, Joo GB, Westvik AB, Kierulf P. Net inflammatory capacity of human septic shock plasma evaluated by a monocyte‑based target cell assay: Identification of interleukin‑10 as a major functional deactivator of human monocytes. J Exp Med 1996;184:51‑60.

Natanson C, Esposito CJ, Banks SM. The sirens' songs of confirmatory sepsis trials: Selection bias and sampling error [editorial]. Crit Care Med 1998;26:1927‑1931.

Reinhart K, Wiegand‑Lohnert C, Grimminger F, Kaul M, Withington S, Treacher D, Eckart J, Willattts S, Bouza C, Krausch D, Stockenhuber F, Eiselstein J, Daum L, Kempeni J. Assessment of the safety and efficacy of the monoclonal anti‑tumor necrosis factor antibody‑fragment, MAK 195F, in patients with sepsis and septic shock: A multicenter, randomized, placebo‑controlled, dose‑ranging study. Crit Care Med 1996;24:733‑742.

IL‑6 and Tumor Necrosis Factor Levels as Markers of Response in Sepsis Trials

Although elevated circulating IL‑6 levels have been shown to correlate with poor outcome in septic patients, it has been more difficult to show any relation between IL‑6 and response to therapy. The INTERSEPT and NORASEPT II trials examined the utility of a murine antitumor necrosis factor (anti‑TNF) alpha monoclonal antibody in patients with septic shock. In neither study was any predictive value associated with IL‑6 levels and response to anti‑TNF therapy. Similarly, a p55 TNF receptor fusion protein was studied in two clinical trials of patients with severe sepsis with or without hypotension. Although there was a significant relationship in both studies between IL‑6 levels and outcome, there was no predictive value apparent for response to anti‑TNF therapy and IL‑6 levels. In contrast, a small study using Fab fragments of a murine monoclonal antibody showed a linear relationship between dose of this anti‑TNF therapy and improved survival, but only in patients with IL‑6 levels greater than 1,000 pg/mL. Such results were unable to be confirmed in a larger European study. However, a large North American study of more than 2,000 patients aimed at further examining the relationship between IL‑6 levels and outcome to this anti‑TNF therapy was recently completed, and results should be available shortly. Circulating levels of TNF are present in only a minority of patients with severe sepsis or septic shock. However, in the NORASEPT II trial, those patients with detectable plasma TNF alpha levels seemed to have better response to the anti‑TNF antibody than patients without such elevations in circulating TNF.

Abraham E, Marshall JC. Sepsis and mediator‑directed therapy: Rethinking the target population. Mol Med Today 1999;5:56‑58.

Tracey KJ, Abraham E. From mouse to man: Or what have we learned about cytokine‑based anti‑inflammatory therapies. Shock 1999;11:224‑225.

Cytokine Balance in Acute Respiratory Distress Syndrome: Implications for Detecting Acute Lung Injury

Acute respiratory distress syndrome (ARDS) is characterized by an intense inflammatory response in the lungs that begins before ARDS is clinically evident. Markers of inflammation in blood and lungs were among the first measurements made to predict the onset and outcome of ARDS. Despite initial studies in small samples, single cytokine markers in blood and lung fluids are not consistently predictive. The biological activity of individual cytokines is determined by the balance between cytokines and their naturally occurring inhibitors and by other factors, such as binding to tissue matrix. In the Seattle ARDS Specialized Center of Research program, we have prospectively studied 25 patients at risk for ARDS and 45 with established ARDS, using serial bronchoalveolar lavage. Proinflammatory cytokines and chemokines are detectable in the lungs of patients at risk, increase at the onset of ARDS, and decline with time. For tumor necrosis factor alpha, IL‑1alpha, and IL‑6, the molar concentrations of naturally occurring inhibitors (sTNFRI and II, IL‑1RA, sIL‑6R and others) exceed those of the ligands at all times by tenfold or more. High concentrations of anti‑IL‑8 IgG and alpha‑2‑macroglobulin, which binds IL‑8, also are present. The only cytokine that increases with time in ARDS is migration inhibitory factor, a naturally occurring antagonist of the inhibitory effects of cortisol on macrophage cytokine production. Neither the cytokines nor their inhibitors were strong predictors of outcome. Thus, cytokine balance complicates the use of single cytokine measurements as predictors of ARDS. Markers of the effects of inflammation on the structural components of the alveolar wall may prove to be more useful in predicting the onset or outcome of ARDS.

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Goodman RB, Strieter RM, Steinberg KP, Milberg JA, Martin DP, Maunder RJ, Kunkel SL, Walz A, Hudson LD, Martin TR. Inflammatory cytokines in patients with persistence of the acute respiratory distress syndrome. Am J Respir Crit Care Med 1996;154:602‑611.

Martin TR. Cytokines and the acute respiratory distress syndrome (ARDS): A question of balance. Nat Med 1997;3:272‑273.

Miller EJ, Nagao S, Griffith D, Maunder RJ, Martin TR, Wiener‑Kronish JP, Matthay MA, Sticherling M, Chrostophers E, Cohen AB. Elevated levels of NAP‑1/interleukin‑8 are present in the airspaces of patients with the adult respiratory distress syndrome and are associated with increased mortality. Am Rev Respir Dis 1992;146:427‑432.

Pittet JF, Mackersie R, Martin TR, Matthay MA. Biological markers of acute lung injury: Prognostic and pathogenetic significance. Am J Respir Crit Care Med 1997;155:1187‑1205.

Suter PM, Suter S, Girardin E, Roux‑Lombard P, Grau GE, Dayer JM. High bronchoalveolar levels of tumor necrosis factor and its inhibitors, interleukin‑1, interferon, and elastase, in patients with adult respiratory distress syndrome after trauma, shock, or sepsis. Am Rev Respir Dis 1992;145:1016‑1022.

The cause of death in most intensive care unit (ICU) patients is multiple organ dysfunction or failure (MOD). Life support is increasingly effective (at least in the short term). Well‑established methods exist for outcome prediction (e.g., APACHE, SAPS, MPM), but there are no well‑established methods for systematically quantifying morbidity (or severity of illness). We hypothesized that standardized assessment of organ dysfunction can be used as a tool to measure important clinical morbidity in clinical trials and clinical practice. Methods included development of standard definitions through a series of consensus conferences (Antioxidants in ARDS Study Group, Chicago, July 1993; Acute Lung Injury Specialized Center of Research Coordination Group, Bethesda, September 1993; Sepsis Round Table Group, Brussels, March 1994). The selection criteria for MOD assessment variables were that they be (1) simply and easily measured, (2) useful in heterogeneous groups of patients, (3) reflective of specific organ function, (4) unaffected by therapeutic interventions that may appear to but do not restore organ function, (5) a continuous variable, (6) abnormal in only one direction, and (7) correlative with increasing mortality. Results will be presented at the conference. MOD assessment is simple and easy to apply and describes ICU morbidity in a clinically meaningful manner and when combined with OFF (organ failure‑free) day analysis, avoids the confounding effect of high mortality rates. Such a measure would be a useful standard measure of outcome in clinical trials in critically ill subjects. It is likely to be a more sensitive and specific outcome variable than mortality in clinical trials in the critically ill.

Arons M, Wheeler AP, Bernard GR, et al. Developing an improved lung injury score. Am J Respir Crit Care Med 1997;155(4):A584.

Arons M, Wheeler AP, Hudson LD, Carroll FE, Morris PM, Summer W, Schein R, Swindell B, Steinberg K, Wright P, Fulkerson W, Bernard GR. Chest x‑rays, gas exchange measurements and the lung parenchymal injury score in patients with sepsis induced lung injury. Am J Respir Crit Care Med 1996;153(4):A123.

Bernard GR, Artigas A, Brigham KL, Carlet J, Falke K, Hudson L, Lamy M, LeGall JR, Morris A, Spragg R, Consensus Committee. Report of the American‑European Consensus Conference on Acute Respiratory Distress Syndrome (Part I): Definitions, mechanisms, relevant outcomes, and clinical trial coordination. Simultaneous publication in J Crit Care 1994;9(1):72‑81; Am J Respir Crit Care Med 1994;149:818‑824; Intensive Care Med 1994;20:225‑232.

Bernard GR, Doig G, Hudson LD, Lemeshow S, Marshall JC, Russell J, Sibbald W, Sprung CL, Vincent JL, Wheeler AP. Quantification of organ failure for clinical trials and clinical practice. Am J Respir Crit Care Med 1995;151(4):A323.

Hebert PC, Russell JA, Drummond AJ, Singer J, Bernard GR. A simple multiple system organ failure scoring system predicts mortality of patients who have sepsis syndrome. Chest 1993;104:230‑235.

Marshall JC, Cook DJ, Christou NV, Bernard GR, Sprung CL, Sibbald WJ. The multiple organ dysfunction (MOD) score: A reliable descriptor of a complex clinical outcome. Crit Care Med 1995;23(10):1638‑1652.

Singer J, Russell J, Drummond A, Fulkerson W, Hudson L, Schein R, Steinberg K, Summer W, Wheeler A, Wright P, Bernard GR. Multiple organ dysfunction evaluation system (MODES) as an outcome of human sepsis syndrome: correlation with mortality. Am J Respir Crit Care Med 1996;153(4):A833.

Critical care risk evaluation systems that utilize demographic and biochemical parameters have been proposed as a means to direct some therapeutic decisions or to evaluate responses to therapy. The refinement of additional disease or condition‑specific biomarkers to enhance therapeutic decisions has great appeal but lacks prospective investigation. In theory, such a biomarker should be definitively altered by the prevailing clinical condition and responsive to intervention. Such markers should also be attainable in real time, be cost effective, and exhibit a high degree of sensitivity and specificity with respect to clinical outcome (surrogate endpoint). The function of solid organs is currently assessed by biochemical or clinical parameters that do not provide insight into the competence of the innate and acquired immune systems. Toward this end, recent efforts to quantify soluble inflammatory mediators or their respective soluble or cell‑associated receptors suggest that such an approach is clinically feasible and potentially useful as a surrogate endpoint of disease severity in critically ill patients. It remains to be established that such markers are responsive to novel therapies. Results of these studies and the prospects for development of biomarkers of immune function will be discussed.

Calvano SE, Coyle SM, Barbosa KS, Barie PS, Lowry SF. Multivariate analysis of nine disease‑associated variables for outcome prediction in patients with sepsis. Arch Surg 1998;133:1347‑1350.

Hubl W, Wolfbauer G, Streicher J, Andert S, Stanek G, Fitzal S, Bayer PM. Differential expression of tumor necrosis factor receptor subtypes on leukocytes in systemic inflammatory response syndrome. Crit Care Med 1999;27:319‑324.

Patients with sepsis are not a homogeneous population (Parsons and Moss 1996; Abraham et al., in press). They frequently have preexisting/comorbid conditions, including alcohol abuse and diabetes, that contribute to outcome and influence biomarker measurements. In 351 patients at risk for acute respiratory distress syndrome (ARDS), we found that the incidence of ARDS was 43 percent in those with a history of alcohol abuse compared with 22 percent for those who were not alcoholic (Moss et al. 1996). The effect was most pronounced in the cohort of septic patients (n=109) in which the incidence of ARDS was 52 percent in the alcoholics compared with 20 percent. In 113 patients with septic shock, we found that the incidence of ARDS was 25 percent for those with diabetes (n=32) compared with 47 percent for those without (Moss et al. 1997). Alcohol abuse and diabetes also affect the measurements of biomarkers. ICAM‑1 levels are decreased in the circulation of septic patients with a history of alcohol abuse compared with septic patients without alcohol abuse (Moss et al. 1998), and patients at risk for and with diabetes have increased levels of ICAM‑1 in their circulation (Lampeter et al. 1992; Roep et al. 1994). Numerous other preexisting/comorbid conditions, including renal failure, liver disease, age, gender, and drug abuse—which could also influence clinical outcomes and biomarker measurements—remain to be investigated.

Abraham E, Dinarello CA, Matthay MA, Vincent JL, Cohen J, Opal SM, Glauser M, Parsons PE, Fisher CJ, Repine JE. Consensus conference definitions for sepsis, septic shock, acute lung injury, and ARDS ‑Time for a re‑evaluation. Crit Care Med, in press.

Lampeter ER, Kishimoto TK, Mainolfi EA, Rothlein R, Bertrams J, Kolb H, Martin S. Elevated levels of circulating adhesion molecules in IDDM patients and subjects at risk of IDDM. Diabetes 1992;41:1668‑1671.

Moss M, Bucher B, Moore FA, Moore EE, Parsons PE. Predicting the development of the Adult Respiratory Distress Syndrome: The role of chronic alcohol abuse. JAMA 1996;275:50‑54.

Moss M, Duhon G, Brown LAS, Guidot G. Chronic alcohol abuse is associated with reduced soluble ICAM‑1 levels in patients at risk for the acute respiratory distress syndrome (ARDS). Am J Respir Crit Care Med 1998;157:A682.

Moss M, Steinberg K, Guidot D, Duhon G, Treece P, Wolken R, Hudson L, Parsons PE. Diabetic patients with septic shock have a decreased incidence of the acute respiratory distress syndrome (ARDS). Am J Respir Crit Care Med 1997;155:A503.

Parsons PE, Moss M. Early detection and markers of sepsis. Clin Chest Med 1996;17:199‑212.

Roep B, Hedenthal E, De Vries R, Kolb H, Martin S. Soluble forms of intercellular adhesion molecule‑1 in insulin‑dependent diabetes mellitus. Lancet 1994;343:1590‑1593.

Severity of Infectious Challenge Alters the Effects of Anti‑Inflammatory Agents in Sepsis

Although anti‑inflammatory agents were shown to markedly improve survival in published animal models of sepsis, these agents have had at best only small beneficial effects in clinical sepsis trials. These differing results suggest that factors encountered in clinical trials were not controlled for in preclinical studies. One such factor may be the severity of infectious challenge. Humans were studied in sepsis trials with mortality rates of 30 to 50 percent, whereas published preclinical trials were performed in animal models with 70 to 90 percent mortality rates. We therefore investigated the relationship between control mortality and the effects of anti‑inflammatory agents on survival in animal studies of sepsis we performed as well as in published animal and human trials. Both in our own animal studies as well as those in the literature, there was a significant association between control mortality and the effects of anti‑inflammatory agents on survival. Agents were most beneficial at high mortality rates. As mortality rates decreased, agents became less effective or even harmful. The small beneficial effects of anti‑inflammatory agents in clinical trials, where control mortality was not high, were consistent with findings in animal studies. Thus, severity of infectious challenge may be an important factor altering the effects of anti‑inflammatory agents in sepsis. Anti‑inflammatory agents may be most beneficial in septic patients with a high likelihood of dying. However, these agents may have little effect or be harmful in septic patients with less severe infections associated with a low mortality rate. These findings suggest that effective use of anti‑inflammatory agents in sepsis may not be possible unless reliable markers differentiating the severity of infection in patients can be identified.

cDNA microarray technology promises to become a pivotal tool in understanding the functional genomics of complex diseases (Heller et al.1997). Critically ill or injured patients frequently die of incompletely understood conditions such as septic shock, acute respiratory distress syndrome, and ultimately multiple organ dysfunction syndrome. Activation of host inflammatory pathways causes tissue injury and thereby acts as a major pathogenic mechanism in these syndromes. At a basic level, the clinical and biological manifestations of host responses are determined by quantitative and qualitative changes in gene expression. Therefore, organ injury syndromes might be defined by their associated patterns of altered gene expression. From paired samples of cells or tissues, cDNA microarrays can quantitate relative changes in mRNA levels for thousands of genes simultaneously (Chen et al. 1998). Furthermore, cDNA microarrays can be used to detect genetic polymorphisms that affect outcome and to identify new gene targets for drug development (Ramsay 1998). Because cDNA microarrays generate huge data sets even from relatively simple experiments, difficulties with validating and conceptually handling this quantity of information need to be resolved. Clustering data from genes with shared characteristics, developing software to aid in the interpretation of results, and rapidly making results widely available (Iyer et al. 1999) are some potential approaches to these problems. Serial samples from patients, human or rodent models, and cultured cells could be used to create a public‑domain, functional genomics database relevant to investigating septic shock and multiple organ failure.

Chen JJ, Wu R, Yang PC, Huang JY, Sher YP, Han MH, Kao WC, Lee PJ, Chiu TF, Chang F, Chu YW, Wu CW, Peck K. Profiling expression patterns and isolating differentially expressed genes by cDNA microarray system with colorimetry detection. Genomics 1998;91:313‑324.

Heller RA, Schena M, Chai A, Shalon D, Bedilion T, Gilmore J, Woolley DE, Davis RW. Discovery and analysis of inflammatory disease‑related genes using cDNA microarrays. Proc Natl Acad Sci 1997;94:2150‑2155.

Iyer VR, Eisen MB, Ross DT, Schuler G, Moore T, Lee JCF, Trent JM, Staudt LM, Hudson J Jr, Boguski MS, Lashkari D, Shalon D, Botstein D, Brown PO. The transcriptional program in the response on human fibroblasts to serum. Science 1999;283:83‑87.

Biomarkers and Surrogate Endpoints for Anti‑Inflammatory Therapies for Sepsis

Mortality, the endpoint of primary clinical interest in sepsis, occurs in about 30 percent of patients in most trials, usually within a few days to a few weeks. Thus, frequency and rapidity of the endpoint do not give rise to need for a surrogate. However, drug development in this area has been extremely difficult, in part because of the broad diversity of the target population. Sepsis patients differ with regard to many parameters that might influence response to therapy, including site of infection, type of infection, underlying disease, metabolic state, presence of shock, and organ failure. Identifying the optimal target population, dose, regimen, and concomitant therapies could be greatly facilitated by identification of a marker that effectively predicts effects on mortality or other important clinical outcomes. However, given the diversity of pathophysiological processes present during sepsis, one must be cautious about assuming that an agent with a favorable effect on one will not have an unfavorable effect on others. U.S. Food and Drug Administration‑coordinated efforts currently under way to analyze sepsis trials to date and to identify useful markers will be described briefly.