Procalcitonin as a biomarker for predicting bacterial infection in chimeric antigen receptor T‐cell therapy recipients

Abstract Background It is unknown whether serum procalcitonin (PCT) concentration monitoring can differentiate between bacterial infection or cytokine release syndrome (CRS) when chimeric antigen receptor T‐cell (CAR‐T) recipients present with a constellation of signs and symptoms that may represent both complications. Objective The objective of the study was to assess the utility of serum PCT concentrations as a biomarker of bacterial infection in CAR‐T recipients. Study design This single‐center, retrospective, medical record review evaluated patients prescribed CAR‐T therapy until death or 30 days after infusion. Logistic regression modeling determined the association between elevated serum PCT concentrations within 48 h of fever onset and microbiologically confirmed infection. Secondary outcomes included clinically suspected infection, CAR‐T toxicity rates, and broad‐spectrum antibiotic usage. Predictive performance of PCT was assessed by area under the receiver operating characteristic curve (AUC). Results The 98 included patients were a median age of 63 (IQR: 55–69) years old, 47 (48%) were male, and 87 (89%) were Caucasian. Baseline demographics and clinical characteristics were similar between patients with and without a bacterial infection. Serum PCT >0.4 ng/mL within 48 h of fever was significantly associated with a microbiologically confirmed bacterial infection (OR: 2.75 [95% CI: 1.02–7.39], p = 0.045). Median PCT values in patients with and without confirmed infections were 0.40 ng/mL (IQR: 0.26, 0.74) and 0.26 ng/mL (IQR: 0.13, 0.47), respectively. The AUC for PCT to predict bacterial infection was 0.62 (95% CI 0.48–0.76). All patients experienced CRS of some grade, with no difference in CRS severity based on elevated PCT. Broad‐spectrum antibiotics were used for a median of 45% and 23% of days in those with and without confirmed infection, respectively (p = 0.075). Conclusion Elevated serum PCT concentrations above 0.4 ng/mL at time of first fever after CAR‐T infusion was significantly associated with confirmed bacterial infection. Furthermore, rigorous, prospective studies should validate our findings and evaluate serial PCT measurements to optimize antimicrobial use after CAR‐T therapy.


| INTRODUCTION
Chimeric antigen receptor T-cell (CAR-T) therapy represents a major advancement in the management of refractory or relapsed hematologic malignancies. [1][2][3][4] CAR-T therapy is not without toxicities and clinical complexity following administration. [5][6][7][8][9] Cytokine release syndrome (CRS) is a common toxicity following CAR-T therapy that is characterized by high fevers, tachycardia, hypotension, and hypoxia. 5 Bacterial infection is another well-known complication, with particularly high risk during the first 30 days following CAR-T infusion. 6 Bacterial infection and CRS have similar presentations but vastly different management strategies. [10][11][12][13][14] The inability to differentiate between CRS and bacterial infection can lead to unnecessary antibiotic administration. The negative consequences of unnecessary antibiotic prescribing include increased hospital length of stay, increased hospital costs, potential antibiotic-associated Clostridium difficile, secondary infections, and emergence of multidrug-resistant bacteria. 15 Procalcitonin (PCT) is a calcitonin precursor hormone that is upregulated in response to circulating bacteria or endotoxins. 16 Studies have demonstrated that serum PCT concentration monitoring has utility in the diagnosis and management of sepsis and lower respiratory tract infection. [18][19][20][21][22][23] Typically, PCT levels begin rising within 3-4 h of bacterial infection and usually peak between 12 and 36 h. 17 Additionally, serum PCT monitoring has been found to successfully predict infection, particularly bacteremia, in patients with hematological malignancies. [24][25][26][27][28] Lastly, longitudinal serum PCT monitoring can facilitate antibiotic discontinuation without compromising patient safety. 29,30 It is unknown whether serum PCT concentration monitoring can differentiate between bacterial infection or CRS when CAR-T therapy recipients present with a constellation of signs and symptoms that represent either complication. This study sought to evaluate whether serum PCT concentrations could indicate microbiologically confirmed bacterial infection and predict any bacterial infection.

| MATERIALS AND METHODS
This Mayo IRB-approved single-center, retrospective, medical record review evaluated consecutive adult patients diagnosed with B-cell malignancy who received CAR-T therapy between 2018 and 2021 until 30 days after the CAR-T infusion or death. Patients who did not experience a fever after CAR-T infusion or have a serum PCT level within 48 hours of fever onset were excluded.
The primary outcome was the association between elevated serum PCT concentrations within 48 h of fever onset and microbiologically confirmed bacterial infection. Secondary outcomes included the association between elevated PCT and incidence of clinically suspected infection, a comparison of CRS rate and severity in those with and without bacterial infection, and frequency of broadspectrum antibiotic use. Clinically suspected infection was determined by author discretion based on symptomatic presentation and radiographic evidence of infection. CRS was graded according to the American Society for Blood and Marrow Transplantation (ASTCT) consensus guidelines. 5 In addition to serum PCT; ferritin, white blood cell count (WBC), and C-reactive protein (CRP) were assessed as distinguishing laboratory markers indicative of CRS or bacterial infection.
Frequencies and percentages summarized categorical data, and medians and interquartile ranges summarized continuous data. Baseline characteristics were compared between those who did and did not have a bacterial infection using either a chi-squared for Fisher's exact test for categorical data, and the Wilcoxon rank sum test for CRS severity based on elevated PCT. Broad-spectrum antibiotics were used for a median of 45% and 23% of days in those with and without confirmed infection, respectively (p = 0.075).
Conclusion: Elevated serum PCT concentrations above 0.4 ng/mL at time of first fever after CAR-T infusion was significantly associated with confirmed bacterial infection. Furthermore, rigorous, prospective studies should validate our findings and evaluate serial PCT measurements to optimize antimicrobial use after CAR-T therapy.

K E Y W O R D S
antibiotics, antimicrobial stewardship, chimeric antigen receptor, cytokine release syndrome, fever, immunotherapy, infection, procalcitonin, T-cell continuous data. The association between PCT levels and bacterial infection was assessed using logistic regression. Associations were summarized using odds ratios (OR) and 95% confidence intervals (CI). A cut-point analysis was done to determine the optimal cutoff for PCT to distinguish who would be considered high risk for infection. The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) for the new cutoff were assessed. The discrimination of each biomarker was evaluated using the area under the receiver operating characteristic curve (AUC). All analyses were performed using SAS version 9.4 software (SAS Institute Inc.).

| RESULTS
The 98 included patients were a median age of 62.7 (IQR: 54.7-69.0) years, 47 (48%) were male and 87 (88.8%) were Caucasian. Baseline demographics and clinical characteristics were similar between groups except for a lower alkaline phosphate in patients without confirmed bacterial infection ( Table 1). The median (IQR) time to first fever of 4 (2, 6) days from CAR-T cell infusion date into the result section. We have also added the median (IQR) number of 10 (7, 12) cultures drawn as part of the infectious workup during empiric broad-spectrum antibiotic therapy.
All patients experienced CRS of some grade, with no statistically significant difference in CRS severity based on elevated PCT (Table 3). Broad-spectrum antibiotics were used for a median of 33% and 23% of days in patients with and without confirmed bacterial infection, respectively (p = 0.33). The most common sites of microbiologically confirmed bacterial infections were central venous catheters (n = 11), urine (n = 4), and blood (n = 3). Organisms grown from culture were mostly of the Staphylococcus genus (n = 18), including Staphylococcus epidermidis (n = 10), Staphylococcus hominis (n = 2), and Staphylococcus haemolyticus (n = 2), Staphylococcus aureus (n = 1) and coagulase-negative Staphylococcus species not otherwise specified (n = 3). Other organisms detected during infectious work-up included Clostridium difficile (n = 2), Escherichia coli (n = 1), Klebsiella pneumoniae (n = 1), Terrisporobacter glycolicus (n = 1), and Micrococcus luteus (n = 1). Clinically suspected infections were primarily pneumonia (n = 16), intra-abdominal infections (n = 3), or cellulitis (n = 2). There were zero invasive fungal infections during infection workup. Additionally, there were 14 positive viral cultures including one patient with a positive herpes simplex virus swab from the mouth, 6 patients with viral organisms detected in respiratory secretions, and one patient with BK virus positive in the urine. Other viral cultures included transient detection of cytomegalovirus by polymerase chain reaction during routine surveillance blood cultures.

| DISCUSSION
Our study demonstrated that a serum PCT ≥0.4 ng/mL within 48 h of fever in patients who have received CAR-T cell therapy were significantly more likely to have a confirmed bacterial infection compared to those with a serum PCT <0.4 ng/mL. PCT with a cutoff value of 0.4 ng/mL had fair test characteristics; however, clinical relevance was variable with a low PPV and a fair NPV. The predictive performance of PCT for infection was similar to that of ferritin, WBC, and CRP.
Procalcitonin has been studied as a predictor of bacterial infection in many clinical environments and different patient populations and its utility remains controversial. [31][32][33] Previously determined PCT thresholds, diagnostic accuracy, and predictive performance have varied by immune status, disease state, patient population, clinical presentation, outcome studied, and test used. 16,19,20,[25][26][27][28]34,35 The cutoff of 0.4 ng/mL determined in our study differs from the 0.25 ng/mL that is commonly used for community-acquired bacterial pneumonia. 21 Additionally, it is lower than the cutoff value of 1.5 ng/ mL that demonstrated utility in diagnosing bacterial infections in patients undergoing hemodialysis. 36 However, previous studies have suggested that differences of PCT concentrations exist in patients with hematologic malignancies. 24,25,27,28,34,[37][38][39] Interestingly, our PCT value was similar to another study that assessed this biomarker in CAR-T therapy recipients. 40 Differences in study design and outcomes preclude direct comparison; however, one reason that PCT was likely not associated with infection in some other evaluations was the inclusion of viral, fungal, and other infection types; whereas, our outcome focused solely on any bacterial infection, regardless of severity. The increased incidence of Gram-positive organisms as the causative pathogen in our study is likely due to fluoroquinolones prescribed as antibacterial prophylaxis during the period of neutropenia as part of our institutional protocol in accordance with published recommendations. 11 Studies have demonstrated  serum PCT concentrations in patients with a bacterial infection caused by a Gram-positive organism are generally lower than in patients with infections and sepsis due to Gram-negative organisms. 35,41 Furthermore, serum PCT has the ability to distinguish blood contamination from bloodstream infection due to coagulase-negative staphylococci. 26 This is important because these organisms may have clinical significance, particularly when detected in an immunocompromised host. 42,43 The predictive performance of PCT was poor for use as a solitary, independent, diagnostic marker; though, expert consensus recommends it as an adjunct assay in combination with clinical and microbiological assessment. [44][45][46] This result is below what has been found in other studies assessing PCT in patients with malignancy and might reflect the difference in inflammation experienced by patients diagnosed with CRS after CAR T-cell infusion. However, the predictive performance was similar to that of other markers commonly used in clinical practice to support a diagnosis of infection. We feel that the overlap between both clinical and chemical presentations of bacterial infection and CRS is what rendered WBC count and CRP inconsequential for a differential diagnosis specific for infection. Cytopenias are common after lymphodepletion and can be profound and prolonged in up to 46% of CAR-T recipients. 47 Additionally, marked elevations in Creactive protein and ferritin can occur during CRS is not specific for CRS or infection, but is often used to follow inflammation that can result from either diagnosis. 5 Several guidelines recommend utilizing PCT levels plus clinical criteria to guide antibiotic discontinuation. 40,48 CAR-T recipients experience high antimicrobial usage with current recommendations favoring antimicrobial prophylaxis. 11,14,49 Additionally, the high incidence of CRS in CAR-T recipients that is indistinguishable from infection when presenting with fevers during neutropenia compels prompt administration of appropriate broadspectrum antibacterial therapy. 10,11,14 This is evident in our study where broad-spectrum antibiotics were used for a median 23% of hospital days in patients without confirmed infection. This indicates a need for better stewardship that can prevent or minimize antibiotic overuse in this vulnerable patient population to abrogate the many negative consequences that could follow. 50 Limitations include the small sample size, retrospective design, and unblinded nature of data collection. Additionally, baseline PCT values were missing in a substantial amount of our patients. This information should be collected in future studies, though a study in a similar patient population showed a median PCT at baseline that was nearly undetectable, but with a variable range. 40 Third, our study included patients with three different baseline cancer diagnoses and evaluating serum PCT performance independently in patients with lymphoma and multiple myeloma may reveal disease-specific parameters. Fourth, the heterogeneity of our confirmed infections may have impacted performance and focusing on a particular site of infection could demonstrate greater utility in serum PCT measurements. Lastly, PCT values trended over time were also unavailable in the majority of patients, precluding our ability to understand the utility of PCT in assisting with antibiotic de-escalation or discontinuation.
T A B L E 3 Association of serum procalcitonin with bacterial infection and toxicity outcomes.