The ImmuKnow® assay has been examined in clinical trials for its potential use in monitoring immunosuppression medication regimens in solid organ transplant patients.
Assessment of a diagnostic technology typically focuses on three analyses:
- Analytic validity including comparison to a “gold-standard” test and test/re-test reliability;
- Clinical validity including sensitivity, specificity, positive and negative predictive value in appropriate populations of patients; and
- Clinical utility that is demonstration that the information from the diagnostic test results in improved health outcomes.
The sensitivity of a test is the ability to detect disease when the disease is present (true positive), while specificity indicates the ability to detect patients who do not have the disease (true negative). Evaluation of clinical validity, therefore, requires independent assessment by two methods in a population of patients suspected of having a disease but not all of whom do have the disease/disorder. Additionally, demonstration of the clinical utility of the ImmuKnow assay would require specifying abnormal levels prior to testing an immunosuppressed patient population, making treatment decisions based on the assay results, and documenting decreased morbidity and/or mortality (such as improved transplant organ survival and/or reduced infectious complications) following these treatment decisions.
In support of Cylex’s application for FDA clearance, Kowalski et al. conducted a multi-center study of 155 apparently healthy adults and 127 solid organ transplant recipients (59% kidney, 34% liver, 2% pancreas, 5% simultaneous kidney and pancreas). Immunosuppressive therapies among the transplant recipients were not limited and included muromonab (lymphocyte-depleting antibody, OKT3), anti-thymocyte globulin, calcineurin inhibitors (cyclosporine, tacrolimus), steroids and mycophenolate mofetil, a purine synthesis inhibitor. ImmuKnow assays were performed < one month to > four years after transplant. Additional details on testing were not specified. Ninety-two percent of the transplant patients had CD4+ ATP levels < 525 ng/ml, while 94% of apparently healthy controls had CD4+ ATP values > 225 ng/ml.
The authors concluded that this defines three zones of patients’ immune response:
- Adenosine triphosphate(ATP) level < 225 ng/ml indicates that the patient’s circulating immune cells are showing a low response to phytohemagglutinin(PHA) stimulation and suggests that the patient may be at increased risk of infection;
- ATP level > 525 ng/ml indicates that the patient’s circulating immune cells are showing a strong response to PHA stimulation and suggests that the patient may be at increased risk of transplant rejection;
- A moderate ATP level (i.e., between 225 and 525 ng/ml) represents a proposed ideal response to PHA stimulation.
ATP level was not correlated with CD4+ T-cell count.
These transplant recipients were included in a follow-up manufacturer-supported study. The ImmuKnow assay was completed on 504 immunosuppressed transplant recipients (48% kidney, 30% liver, 17% heart, and 5% small bowel) within 30 days after an episode of infection or rejection. Because only 5% of patients with ATP levels between 130 ng/ml and 450 ng/ml demonstrated adverse events (either infection or rejection), the authors propose this as the target range for ATP level in immunosuppressed transplant recipients. Note that this analysis yielded different ATP threshold levels for infection risk and rejection risk than those developed in the earlier study and cited in the product insert. Further, a 2005 manufacturer-supported study of 37 stable pediatric kidney transplant recipients (mean age = 11.1 years) suggests that in children < 12 years of age, risk intervals are defined by ATP level > 395 ng/ml for rejection and < 175 ng/ml for infection.
A manufacturer-supported single-center study assessed 20 small bowel transplant recipients (70% isolated small bowel, 10% multi visceral, 10% modified multi visceral, 10% simultaneous liver, small bowel and pancreas) undergoing tacrolimus tapering per protocol 60-190 days post-transplant. Among eight patients successfully tapered from tacrolimus, 70% of ATP levels clustered in the low range (< 225 ng/ml), with 25% of ATP levels occurring in the moderate range and 5% occurring in the strong range. Incidence of infection was not reported. Twelve unstable transplant recipients (requiring addition of steroid or OKT3) showed ATP levels with 30% in the low range, 43% in the moderate range, and 27% in the strong range. This study is often described as using ImmuKnow assay results to guide tacrolimus dosing. However, adjustments to the tapering protocol were determined primarily by histological examination of biopsy results, which correlated with ATP levels as described for the unstable group.
The relationship between low post-transplant ATP levels (< 225 ng/ml) and recent infection in 57 immunosuppressed adult lung transplant recipients was assessed by Bhorade et al. One hundred forty-three ImmuKnow assays were performed at routine clinic visits when each patient was on a stable dose of tacrolimus. Fifteen patients developed infections (bacterial or fungal pneumonia, cytomegalovirus infection); 14 of these (93%) had ATP levels < 225 ng/ml at the time of their infections. Among the 42 non-infected patients, 16 (38%) had ATP levels < 225 ng/ml. Without comparing post-infection ATP levels with pre-infection ATP levels, it is not possible to draw conclusions about whether a low ATP level contributes to or results from the development of infection.
Of note in this study was the finding that “African American race was an independent predictor of decreased ImmuKnow assay levels that remained significant with stepwise multiple regression analysis. Interestingly, African American lung transplant recipients received higher doses of tacrolimus that led to similar tacrolimus trough levels, but had significantly decreased immunoassay levels.” The authors cite data indicating “African Americans who have undergone renal transplantation require a 37% mean higher dose of tacrolimus to achieve similar blood concentrations” and conclude that African-Americans “may be over-immunosuppressed based on the concurrently obtained ImmuKnow assay.” Because the authors did not report the incidence of infection among African American patients with ATP level < 225 ng/ml, it is not possible to conclude whether these findings are clinically significant.
Two studies found no correlation between ATP levels as determined by the ImmuKnow assay and outcomes in cardiac transplant recipients. Rossano et al. studied 83 pediatric patients (median age, 4.9 years) undergoing heart transplant. ImmuKnow assays were performed at routine follow-up visits from three months to over five years after transplant. There were 26 episodes of acute rejection, 20 (77%) of which were cell-mediated, and the remainder were humoral rejection. There were 38 infections. No difference in ATP levels as measured by ImmuKnow assay was detected between patients with or without acute rejection or with or without infection. Further, the manufacturer’s reported risk ranges for rejection (ATP level > 525 ng/ml) or infection (ATP level < 225 ng/ml) were not predictive of rejection or infection respectively. As noted above, however, it may be that pediatric patients’ risks for post-transplant infection and rejection correspond to different ATP levels.
Gupta et al. studied 125 adult heart transplant recipients, the majority of whom underwent ImmuKnow assay testing > one year post-transplant. There was no apparent correlation between ATP level and rejection (n=3). For seven patients who developed infection, the median ATP level was 267ng/ml and did not differ from the median ATP level in 104 patients who did not develop infection (282 ng/ml). There was a significant correlation between ATP level and white blood cell count, but not between ATP level and absolute lymphocyte count, suggesting that non-lymphocytes also may influence the ATP response. This idea is supported by a 1994 study of CD4+ T-cell responsiveness to three stimulants (including phytohemagglutinin) in HIV+ patients. The authors suggest that assays performed in clinical laboratories should profile immunoregulatory cytokines (e.g., interleukin-2) which modulate the complex interplay between cellular and humoral immune mechanisms.
The studies cited above report ImmuKnow assay results obtained after transplant. Reinsmoen and colleagues studied 126 kidney transplant recipients to determine whether pre-transplant immune parameters (ATP level as well as human leukocyte antigen (HLA) mismatch, HLA-specific antibodies, and IFN-gamma precursor frequencies to donor or third-party cells) were associated with post-transplant early acute rejection, unstable creatinine course, and poor graft outcome. The mean pre-transplant ATP level of recipients who had no clinical reason for a biopsy was significantly different from that of recipients who had biopsy-proven AR at any post transplant time point up to 36 months (285.3 ± 143.2 vs. 414.3 ± 138.5 ng/ml). Recipients who underwent biopsy but had no diagnosis of acute cellular or antibody-mediated rejection had an intermediate mean value of 333.7 ± 156.3 ng/ml. Pre-transplant ATP levels were also significantly higher for recipients with early (< 90 days) unstable creatinine levels, a significant predictor of early acute rejection, than for recipients with stable creatinine values (362.8 ± 141.2 vs. 283.4 ± 146.4 ng/ml). Post-hoc analysis using a cutoff ATP level of 375 ng/ml revealed that recipients with pre-transplant ATP > 375 ng/ml were significantly more likely to experience acute rejection (OR=3.67, 95% confidence interval, 1.195, 11.201). The immune parameters were not used to guide modifications of the immunosuppression protocol. Graft survival and incidence of infection was not reported in this study. In their discussion, Reinsmoen et al. suggest that CD4+ T-cell production of ATP in response to PHA stimulation may depend upon the immunosuppressive medications employed: “Induction immunosuppression can nonspecifically reduce precursor frequencies; however, not all T cells are depleted equally. T-cells with a memory phenotype [i.e., CD4+] appear to be less susceptible to depletion than naïve T-cells. Results from studies of alemtuzumab induction therapy have shown that mature T-cells are profoundly depleted, with memory T-cells, B cells, and monocytes depleted to a lesser degree. Further, the recipients in these trials often experienced reversible rejection episodes that were characterized by a predominant monocyte, not lymphocyte, infiltrate. Our results suggest the induction therapy may have had more profound negating effect on the population of cells detected by the ELISPOT [i.e., interferon-gamma producing memory T-cells] but not the CD4+ population of cells detected by the ATP synthesis assay. The association seen between high pre-transplant ATP levels and acute rejection, as seen in this study, may be immunosuppression protocol dependent. These results suggest the relevance of the immune parameters studied may be influenced by the immunosuppression protocol employed.”
The American Society of Transplantation (AST) has published recommendations for the screening, monitoring and reporting of infectious complications in immunosuppression trials of organ transplant recipients. These recommendations define relevant infectious complications to be included in the reporting of immunosuppression trials and recommend specific laboratory monitoring and surveillance methods. The immune cell function assay is not included in these recommendations.
ClinicalTrials.gov lists five studies assessing the ImmuKnow assay’s ability to predict or monitor the development of infection or rejection in solid organ and hematopoietic stem cell transplant recipients and in multiple sclerosis patients. None of the studies includes modifications of immunosuppressive medication regimens based on ATP level.
Without clinical trials demonstrating improved patient outcomes, specifically, reduced incidence of infection, rejection and adverse medication-related effects as a direct consequence of ImmuKnow assay results and subsequent treatment, the evidence is insufficient to permit conclusions concerning the effect of this procedure on health outcomes. Therefore, the ImmuKnow cell function assay is considered experimental, investigational and unproven.
Israeli et al. studied 50 cardiac transplant recipients. 327 samples were acquired and reviewed. Results noted that “Longitudinal monitoring of Immuknow levels through serial testing proved to be a reliable method for individual patient immune management.” The authors concluded “Immunknow assay reliably reflects the cellular immune function of heart transplantation patients, thereby supporting the immune monitoring and management of these patients. Serial longitudinal Immuknow monitoring allows immune management of therapy according to the individual patient’s immune status.”
Xue et al. identified levels of functional immunity in liver transplant patients as measured by the ImmuKnow assay and addressed its application in monitoring posttransplant infection risk. Results that the authors reported were for infectious liver transplant recipients. The average ImmuKnow assay was 128 + or – 84ng/ml. This assay level was significantly lower (P<0.05) than that found in stable liver transplant recipients (305 + or- 149ng/ml). The author’s noted in their conclusion that because liver transplant recipients with infection were found to have lower ImmuKnow adenosine triphosphate levels, immune cell functional assay provided a new means of monitoring posttransplant infection.
Kobashigawa et al. studied 296 heart transplant recipients of which 864 immune monitoring assays were obtained. The results reported by the author noted that during infection, the average immune monitoring score was significantly lower than during steady state. Conclusions noted by the authors were “The non–invasive immune monitoring test appears to predict infectious risk in heart transplant patients.”
The above mentioned studies included solid organ transplantation recipients. These studies were able to demonstrate a relationship between immune monitoring assay levels and the recipients’ immune function. ImmuKnow assay provides a valuable instrument in the supervision and monitoring of the transplant recipients’ immune condition. Thus, the coverage statement is changed to indicate use of an immune cell function assay to monitor and predict immune function after solid organ transplantation maybe considered medically necessary.
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