Pentaglobin® Efficacy in Reducing the Incidence of Sepsis and Transplant-Related Mortality in Pediatric Patients Undergoing Hematopoietic Stem Cell Transplantation: A Retrospective Study

The 12-month mortality rate in patients undergoing hematopoietic stem cell transplantation (HSCT) remains high, especially with respect to transplant-related mortality (TRM), which includes mortality due to infection complications through the aplasia phase. The aim of this study was to determine whether the administration of Pentaglobin® could decrease TRM by lowering sepsis onset or weakening sepsis through the aplasia phase. One hundred and ninety-nine pediatric patients who had undergone HSCT were enrolled in our retrospective study. The patients were divided into two groups: the Pentaglobin group, which had received Pentaglobin® in addition to the standard antibiotic treatment protocol established for the aplasia phase, and the Control group, which received only the standard treatment. As compared to the control group outcome, Pentaglobin® led to a significant decrease in the days of temperature increase (p < 0.001) and a reduced infection-related mortality rate (p = 0.04). In addition, the number of antibiotics used to control infections, and the number of antibiotic therapy changes needed following first-line drug failure, were significantly lowered in the Pentaglobin group as compared to the control group (p < 0.0001). With respect to the onset of new infections following the primary infection detected, the Pentaglobin group showed a significant reduction for bacterial events, as compared to the control group (p < 0.03). Pentaglobin® use in patients undergoing HSCT seems to produce a significant decrease in infection-associated TRM rate.


Introduction
Hematopoietic stem cell transplantation (HSCT) is the most efficient consolidation therapy in some hematologic malignancies such as acute lymphoblastic leukemia and acute myeloid leukemia. HSCT is also a potential therapy for patients with solid tumors, genetic, hematological and metabolic disorders, and primary immunodeficiency diseases [1][2][3][4]. Nevertheless, HSCT results in a variety of severe complications responsible for a high rate of morbidity and mortality in transplant recipients [5].
In recent years, progress has been achieved in transplantation medicine with respect to high-resolution donor-recipient human leukocyte antigen (HLA) matching, conditioning regimens Data were analyzed with respect to a variety of demographic and clinical variables, including sex, age in years, underlying disease, pre-transplant immunity status (namely number of lymphocytes and level of serum immunoglobulins), conditioning regimen, and donor type. In addition, the following information was collected for all patients: type of infection, number of days with temperature rise ≥37.3 °C (99.1 °F), number of fever days, duration of Pentaglobin ® administration, duration of antibiotic treatment, number of antibiotics used, number of antibiotics changed because of treatment failure and acute GVHD onset (any grade).
The primary outcome evaluated was the possible difference in TRM in both groups after a sixmonth follow-up. Secondary outcomes included the comparison between the two groups in the number of fever days, number of infectious events, duration, and number of antibiotics used to control every single event.

HSCT Procedure
All patients who underwent allogeneic HSCT were treated according to standard myeloablative protocols. In patients over two years of age with acute lymphoblastic leukemia (LLA), the myeloablative conditioning regimen preceding allogeneic HSCT was based on total-body irradiation (TBI), while in the remaining cases, a busulfan-based conditioning regimen was used. In both cases, conditioning also included high-dose cyclophosphamide (1800 mg/m 2 for two consecutive days). In the case of matched unrelated donors, haploidentical or sibling donors, and patients with hemoglobinopathy, rabbit anti-thymocyte globulin (ATG) was used. GVHD prophylaxis was performed with calcineurin-inhibitor alone or associated with mycophenolate mofetil and Data were analyzed with respect to a variety of demographic and clinical variables, including sex, age in years, underlying disease, pre-transplant immunity status (namely number of lymphocytes and level of serum immunoglobulins), conditioning regimen, and donor type. In addition, the following information was collected for all patients: type of infection, number of days with temperature rise ≥37.3 • C (99.1 • F), number of fever days, duration of Pentaglobin ® administration, duration of antibiotic treatment, number of antibiotics used, number of antibiotics changed because of treatment failure and acute GVHD onset (any grade).
The primary outcome evaluated was the possible difference in TRM in both groups after a six-month follow-up. Secondary outcomes included the comparison between the two groups in the number of fever days, number of infectious events, duration, and number of antibiotics used to control every single event.

HSCT Procedure
All patients who underwent allogeneic HSCT were treated according to standard myeloablative protocols. In patients over two years of age with acute lymphoblastic leukemia (LLA), the myeloablative conditioning regimen preceding allogeneic HSCT was based on total-body irradiation (TBI), while in the remaining cases, a busulfan-based conditioning regimen was used. In both cases, conditioning also included high-dose cyclophosphamide (1800 mg/m 2 for two consecutive days). In the case of matched unrelated donors, haploidentical or sibling donors, and patients with hemoglobinopathy, rabbit anti-thymocyte globulin (ATG) was used. GVHD prophylaxis was performed with calcineurin-inhibitor alone or associated with mycophenolate mofetil and prednisone, as previously described [13]. All patients who underwent autologous HSCT received myeloablative conditioning regimen according to the current protocols based on the underlying disease. Disease risk was defined according to diagnosis and disease stage [14]. Supportive care for GVHD and infectious disease prophylaxis, mucositis, and veno-occlusive disease (VOD) did not substantially change during the time reported in this study.

Pentaglobin ® Administration
Patients included in the study were divided into two groups. Those who had received Pentaglobin ® , in addition to the standard antibiotic treatment protocol established for the aplasia phase, were identified by accessing pharmacy records. Only those whose first cycle of Pentaglobin ® administration started within 12 h from first body temperature rise ≥37.3 Celsius degrees ( • C, 99.1 • F) were included in the Pentaglobin group. Patients who received started Pentaglobin ® administration more than 12 h after their first temperature rise were excluded from the study. Pentaglobin ® was administered at a dose of 5 mL/kg/day in continuous infusion for three days in most cases. Pentaglobin ® administration was shortened in case of neutrophil engraftment with complete resolution of infection symptoms or extended if symptoms persisted in patients with severe mucositis or documented infection. The second group (the Control group) underwent only the standard antibiotic treatment protocol for the aplasia phase which consisted in the use of a third-generation cephalosporin plus aminoglycoside, as first-line therapy; despite first-line empirical antibiotic therapy, patients who remained febrile after 48 h started vancomycin; whereas, if blood culture results were available, a specific antibiotic treatment was undertaken.

Statistical Analysis
Quantitative variables were reported as median value and range or using the median and interquartile range, whereas categorical variables were expressed as absolute value and percentage. Demographic and clinical characteristics of patients were compared using the chi-square test or Fisher's exact test for categorical variables, whereas the Mann-Whitney rank-sum test or the Student's t-test were used for continuous variables; in addition, we performed proportion tests to compare categorical and continuous variables, as appropriate. The primary endpoint, overall survival, and event-free survival were calculated according to the Kaplan-Meier method. Comparisons between different overall survival and event-free survival probabilities were performed using the log-rank test, whereas multivariate analysis was performed using logistic regression in order to adjust the association of overall survival with clinical and demographic variables. p < 0.05 was considered to be statistically significant, and statistical analysis was performed using R statistical software (R version 3.5.2, 2018 The R Foundation for Statistical Computing, Vienna, Austria).

Study Population
Our cohort consisted of 199 patients: the Pentaglobin group included 95 patients, whereas the control group consisted of 104 patients. The baseline patient characteristics of the 199 patients are summarized in Table 1.
Pre-transplant immunological status of patients shows a statistically significant difference in baseline lymphocyte count between the two study groups (p < 0.0001). In contrast, we did not find significant differences between the baseline IgG and IgM values comparing both groups. These data are displayed in Figure 2. MCHT, myeloablative chemotherapy; TBI, total body irradiation; SD, standard deviation. * Disease stage was defined according to previously published classification. This classification is applied to patients with acute leukemia and myelodysplastic syndrome only. 14.
Pre-transplant immunological status of patients shows a statistically significant difference in baseline lymphocyte count between the two study groups (p < 0.0001). In contrast, we did not find significant differences between the baseline IgG and IgM values comparing both groups. These data are displayed in Figure 2  In the Pentaglobin group, the Pentaglobin ® treatment was started when the white blood cell count dropped below 100/μL in all patients. The mean IgM serum concentration was 17.1 mg/dL with ± 11.4 mg/dL of standard deviation (SD) at the start of Pentaglobin ® treatment. The mean onset of Pentaglobin ® infusion was 6.0 days, with ± 3.9 SD after transplantation, and the mean duration of the In the Pentaglobin group, the Pentaglobin ® treatment was started when the white blood cell count dropped below 100/µL in all patients. The mean IgM serum concentration was 17.1 mg/dL with ± 11.4 mg/dL of standard deviation (SD) at the start of Pentaglobin ® treatment. The mean onset of Pentaglobin ® infusion was 6.0 days, with ± 3.9 SD after transplantation, and the mean duration of the treatment was 3.7 days ± 1.2 SD. We compared the differences of sepsis biomarkers, such as C-reactive protein (CRP) and procalcitonin, in both groups, evaluated at mean onset of Pentaglobin ® use, and our analysis did not show statistically significant differences. The mean serum CRP concentration (normal range <0.5 mg/dL) was 1.8 mg/dL ± 2.4 SD in the Pentaglobin group versus 2.3 mg/dL ± 2.6 SD in the control group (p = 0.1705). Regarding the mean serum procalcitonin levels observed on the same day, they were within the normal range (<0.5 µg/L) in both groups.

Primary Outcome: Six-Month Survival Rate
After a six-month follow-up period, 23 children (24%) had died in the Pentaglobin group, as compared to 34 patients (33%) in the control group. These data included deaths for primary disease recurrence, which were 47.8% versus 32.4%, and TRM, which were 52.2% versus 67.6%, respectively, in the Pentaglobin Group and the control group (Table 2). A proportion test showed no statistically significant difference between study groups with respect to TRM, save for infections among causes of TRM (p = 0.04). The curve obtained by Kaplan-Meier survival analysis showed no statistically significant difference in the six-month overall survival (OS) rate between the study groups ( Figure 3).
in the Pentaglobin Group and the control group (Table 2). A proportion test showed no statistically significant difference between study groups with respect to TRM, save for infections among causes of TRM (p = 0.04).
The curve obtained by Kaplan-Meier survival analysis showed no statistically significant difference in the six-month overall survival (OS) rate between the study groups ( Figure 3). In contrast, Kaplan-Meier survival analysis calculated for deaths due to infectious complications showed a statistically significant difference (p = 0.006) (Figure 4).  Finally, a difference in OS rate after six-month follow-up between the two study groups was evident when excluding mucositis and enteritis among the causes of death, even if statistical significance was not achieved (p = 0.061) ( Figure 5). Finally, a difference in OS rate after six-month follow-up between the two study groups was evident when excluding mucositis and enteritis among the causes of death, even if statistical significance was not achieved (p = 0.061) ( Figure 5). Finally, a difference in OS rate after six-month follow-up between the two study groups was evident when excluding mucositis and enteritis among the causes of death, even if statistical significance was not achieved (p = 0.061) ( Figure 5).

Secondary Outcomes
Box-plot analysis showed a statistically significant difference between the study groups for the number of days with body temperature both for ≥37.3 °C (p < 0.001) and ≥38 °C (p < 0.001).
Statistically significant differences between two study groups were found in the number of antibiotics used concurrently during the same infective episode (p < 0.0001) and the number of changes of antibiotics because of the failure of treatment (p < 0.0001). These box-plot analyses are displayed in Figure 6.

Secondary Outcomes
Box-plot analysis showed a statistically significant difference between the study groups for the number of days with body temperature both for ≥37.3 • C (p < 0.001) and ≥38 • C (p < 0.001).
Statistically significant differences between two study groups were found in the number of antibiotics used concurrently during the same infective episode (p < 0.0001) and the number of changes of antibiotics because of the failure of treatment (p < 0.0001). These box-plot analyses are displayed in Figure 6.
The same analysis performed to compare the number of days of antibiotic therapy and the infective episode recovery rate did not show statistically significant differences. With respect to pathogens identified in blood cultures at sepsis onset, one patient (16.7%) in the Pentaglobin group and three patients (21.4%) in the control group showed Klebsiella pneumoniae, the equal percentage of Pseudomonas aeruginosa was observed, while two patients (33.3%) in the Pentaglobin group and one patient (7.1%) in the control group showed Staphylococcus aureus. Moreover, in the Pentaglobin group, both a case of Brevibacterium and Serratia marcescens were detected. Conversely, Escherichia coli, Enterococcus faecalis, Enterobacter aerogenes (two cases for each pathogen), and one case of Acinetobacter baumannii was identified in the control group.
Also, after the six-month follow-up period, 33 patients (34.7%) in the Pentaglobin group and 39 (37.1%) in the control group presented with new infectious events following the primary ones detected, but without statistically significant difference between the two groups. Moreover, when the analysis was conducted for a subtype of infection (fungal, opportunistic, viral, or bacterial), a statistically significant difference was observed only for bacterial events. Specifically, only one episode (3%) of bacterial infection was reported in the Pentaglobin group versus eight episodes (20.5%) in the control group (p < 0.03).
All secondary outcomes are shown in Table 2. Figure 6. Box plots of secondary outcomes. Box-plot analysis showed a statistically significant difference (p < 0.001) between the study groups for the number of days with body temperature ≥37.3°C (A) and ≥38 °C (B). Statistically significant differences (p < 0.0001) between two study groups were found in the number of antibiotics used (C) and the number of changes of antibiotics because of the failure of treatment (D).
The same analysis performed to compare the number of days of antibiotic therapy and the infective episode recovery rate did not show statistically significant differences. With respect to pathogens identified in blood cultures at sepsis onset, one patient (16.7%) in the Pentaglobin group and three patients (21.4%) in the control group showed Klebsiella pneumoniae, the equal percentage of Pseudomonas aeruginosa was observed, while two patients (33.3%) in the Pentaglobin group and one patient (7.1%) in the control group showed Staphylococcus aureus. Moreover, in the Pentaglobin group, both a case of Brevibacterium and Serratia marcescens were detected. Conversely, Escherichia coli, Enterococcus faecalis, Enterobacter aerogenes (two cases for each pathogen), and one case of Acinetobacter baumannii was identified in the control group.
Also, after the six-month follow-up period, 33 patients (34.7%) in the Pentaglobin group and 39 (37.1%) in the control group presented with new infectious events following the primary ones detected, but without statistically significant difference between the two groups. Moreover, when the analysis was conducted for a subtype of infection (fungal, opportunistic, viral, or bacterial), a statistically significant difference was observed only for bacterial events. Specifically, only one episode (3%) of bacterial infection was reported in the Pentaglobin group versus eight episodes (20.5%) in the control group (p < 0.03).
All secondary outcomes are shown in Table 2.

Discussion
The twelve-month mortality rate after HSCT remains excessive, especially TRM, which is about 10%. Notably, with respect to the mortality rate related to disease progression, many new therapeutic strategies have been developed in recent years, such as cellular therapy with cytokine-induced killer cells (CIK) and chimeric antigen receptor T (Car-T) cell therapy or the use of monoclonal antibody therapy such as inotuzumab, blinatumomab, and brentuximab [15]. However, the introduction of new diagnostic tools and amelioration of the current treatment protocols for infectious complications have so far resulted in little comparable improvement. Hence, it has been difficult to obtain a drop in TRM, especially for mortality related to infections developed through the aplasia phase.
Among the new diagnostic tools and improved treatment protocols is the panfungal real-time PCR assay, allowing amplification of any fungal DNA, significantly reducing the time for diagnosis, compared to standard procedures, such as blood culture and histopathological examination [16]. Another recent lab approach is FilmArray, a fast, accurate, molecular diagnostic testing to detect bacteria, viruses, fungi or parasites in biological samples, while also providing information concerning antibiotic-resistant genes [17].
In addition, therapeutic strategies have been developed as a result of the identification of new antimicrobial molecules, overcoming drug-resistance problems, and through the continuous infusion of time-dependent antibiotics, which improves drug safety and efficacy by reducing administered dosage and the emergence of resistant clones [18,19]. Development and use of therapeutic drug monitoring optimize pharmacological therapy for the individual patient, assisting in planning dosage variation, and managing a possible therapeutic failure [20].
In spite of these improvements, infections remain a common complication in patients undergoing HSCT, especially in the aplasia phase. Accordingly, the main effort of clinicians is to prevent the clinical evolution of infection to sepsis and so to reduce infectious disease-related mortality. Pentaglobin ® , an IgM-enriched immunoglobulin preparation, whose IgM component is responsible for endotoxin antibody activity, seems to be the best therapeutic option to control infectious disease-related mortality in transplant recipients [21]. Several studies have shown a statistically significant mortality rate decrease in cohorts of septic newborns, children, and adolescents undergoing Pentaglobin ® therapy. The advantage of Pentaglobin ® administration has been most evident for infections caused by Gram-negative pathogens [22,23]. To date, however, there are no studies in the available literature about early adjuvant use of Pentaglobin ® for the treatment of patients undergoing HSCT.
The only study previously reported for the pediatric cohort, which compared intravenous immunoglobulin prophylactic use to Pentaglobin ® , was administered to patients within 100 days after allogeneic HSCT. This study showed no differences between two groups concerning neutropenia, number of hospitalization days, total days of fever, number of consecutive detected infectious events, acute GVHD onset, VOD onset, and side effects [24].
On the other hand, in the adult population, Poynton et al. reported prophylactic use of Pentaglobin ® compared to placebo in 63 patients undergoing either autologous or allogeneic HSCT. Their study showed a decreased risk of infection-related mortality in patients who received Pentaglobin ® therapy. Also, endotoxemia was lowered in a statistically significant way in the Pentaglobin ® group [25].
In another study, Jackson et al. tested blood endotoxemia levels in 1000 plasma samples obtained from adult infected transplant patients. The authors concluded that almost 70% of fevers of uncertain origin were associated with high blood endotoxemia and that the endotoxemia level was lower in patients who underwent Pentaglobin ® therapy as compared to the Control group [26].
Our analysis demonstrates that early adjuvant use of Pentaglobin ® through aplasia period leads to statistically significant decrease of days of temperature increase, defined either as T • C ≥ 37.3 or T • C ≥ 38, in addition to a reduced infection-related mortality rate compared to the Control group. These data confirmed previous results obtained by Behre et al., who observed that early use of Pentaglobin ® in neutropenic patients with suspected Gram-negative sepsis-induced blood anti-endotoxin antibodies and decreased the mortality rate [27].
Transplant recipients through aplasia phase present with a lowered level of anti-endotoxin antibody as compared to donors, especially IgM. Therefore, the use of Pentaglobin ® in these patients causes an increased antibody level able to combat high endotoxin levels induced by mucositis, chemotherapy, radiation therapy, and acute GVHD [26].
In addition, our results showed that the Pentaglobin group required fewer antibiotics to control infectious complications and fewer antibiotic therapy changes following first-line drug failure as compared to the control group. Our data support the view that Pentaglobin ® early adjuvant use, in addition to standard protocols of antibiotic treatment, might significantly contribute to infection control.
Our study did not show statistically significant differences in total days on antibiotic treatment, perhaps because, in aplasia phase, the patients usually undergo antibiotic therapy until complete hematological reconstitution is accomplished.
Lastly, our study evaluated a number of new bacterial, viral, fungal, and opportunistic infections following the primary one detected. The results did not show a statistically significant difference between the two groups, save for bacterial ones, reduced in Pentaglobin group, in accordance with previously published data [26,27].
Some limitations of this study should be considered. It is a retrospective, monocentric study, with a relatively short follow-up period for outcomes. However, the relatively large selected sample allowed us to get satisfactory results concerning Pentaglobin ® use for the treatment of bacterial infections in patients undergoing HSCT. Due to these considerations, further investigations, especially randomized controlled trials, could be valuable in defining real drug efficacy in these patients.

Conclusions
Pentaglobin ® early adjuvant use in the treatment of the patients undergoing HSCT, who show temperature rise during aplasia phase, seems to produce a significant decrease in the rate of infection-associated TRM. Therefore, a prospective multicenter randomized controlled trial should be conducted to define real Pentaglobin ® efficacy in patients undergoing HSCT.
Author Contributions: Conception, design of the study and writing, M.N. and C.G.; collection and analysis the clinical data, Z.D. and M.A.; original draft preparation, review and editing, T.L. and B.E. All authors read and approved the final manuscript.

Funding:
The study did not provide any source of funding by the Sponsor.