High Serum miR-361-3p Predicts Early Postdischarge Infections after Autologous Stem Cell Transplantation.

Introduction

Introduced in the 1980s, autologous hematopoietic stem cell transplantation (AHSCT) has become a widely utilized clinical procedure, with approximately 27,600 procedures conducted in Europe and 12,000 in the United States in 2021 [ 1 2 ]. AHSCT is primarily employed for various medical conditions, with plasma cell disorders, mainly multiple myeloma (MM) constituting the majority (55% of AHSCT), followed by non-Hodgkin lymphomas (27%) and Hodgkin lymphoma (HL, 9%) [ 3 ]. Recent investigations have assessed the effectiveness and safety of outpatient AHSCT and/or early discharge strategy, a trend that has gained traction, though it has yet to establish itself as a universally accepted standard practice [ 4 ]. Selecting suitable candidates for outpatient procedures necessitates stringent criteria [ 5 ]. Despite the growing popularity of outpatient AHSCT and/or early discharge strategy, there remains a gap in the availability of clinically validated biomarkers capable of accurately predicting post-AHSCT complications or identifying high-risk individuals. The main adverse events during and after AHSCT include infectious complications [ 6 ]. Even more, according to the Center for International Blood and Marrow Transplant Research, infection was the third most common cause (26%) of mortality of adult patients who died within 100 days post-AHSCT, after the organ failure (31%) and primary disease (31%) [ 2 ]. Notably, infectious complications may occur not only in the first several weeks after the procedure, but some opportunistic infections, including fungal, varicella zoster virus, cytomegalovirus, and Pneumocystis jirovecii infections were recently reported to happen even up to 5 years after the procedure [ 7 ].

There is a shortage of reliable biomarkers that can effectively anticipate complications following AHSCT. Identifying patients susceptible to such complications can potentially mitigate hospitalization costs, reduce hospital stays, and facilitate the informed selection of individuals for inpatient or outpatient procedures. Conventional biomarkers based on cytokine levels or leukocyte subset counts may prove unreliable due to the impact of the AHSCT procedure on these markers, prompting the exploration of alternative biomarker categories.

MicroRNAs (miRNA) constitute a category of short, non-coding RNA molecules, typically spanning 22–24 nucleotides, and emerge as a promising class of biomarkers [ 8 ]. Evidence has substantiated the biological stability of miRNAs within the extracellular space, highlighting their resilience and resistance to easy degradation, positioning them as robust and user-friendly biomarkers [ 9 ]. Despite the advantageous properties of serum miRNAs, their actual usefulness in clinical settings is currently constrained. The growing body of knowledge indicates that miRNAs hold potential for disease diagnosis and offer a reservoir of tools for predicting therapy-related complications or may act as therapeutic agents themselves [ 10 11 12 13 ]. In the field of infection, several miRNAs were assessed as promising features in diagnosis and outcome prediction [ 14 15 16 17 ]. Despite this, the role of miRNAs in the setting of hematopoietic transplant-related infections diagnosis and prediction is unknown.

In this study, we aimed to evaluate the potential role of three selected miRNAs- hsa-miR-155-5p, hsa-miR-320c, and hsa-miR-361-3p, in identifying AHSCT recipients at high risk of infectious events up to 100 days post-transplantation after discharge.

Materials and methods

1. Patients and treatment

Patients who underwent AHSCT at the Department of Hematology and Transplantology, Copernicus Memorial Hospital in Lodz, Poland, between March 2015 and January 2022 consisted of the study cohort. AHSCT eligibility, a diagnosis of hematological malignancy, and an age greater than eighteen years constituted the inclusion criteria. Contraindications to AHSCT were primarily comorbidities as measured by a Hematopoietic Cell Transplantation-Comorbidity Index score of three or higher that were included within the exclusion criteria. MM and lymphoma patients were staged using the International Staging System (ISS) and Ann Arbor classification, respectively. MM patients were treated with high-dose melphalan (200 mg/m 2 ), whereas lymphoma patients received carmustine or bendamustine, etoposide, cytarabine, and melphalan (B(e)EAM). Every patient was administered granulocyte-colony-stimulating factor starting from day +4 post-AHSCT (72 hours post-transplantation) until the point of engraftment. Additionally, during hospitalization, each patient received antimicrobial prophylaxis, which included ciprofloxacin at a dosage of 500 mg twice daily, acyclovir at a dosage of 800 mg twice daily, and fluconazole at a dosage of 400 mg once daily for those identified as high-risk for candidiasis. All patients who underwent AHSCT received vaccines according to local recommendations and international guidelines [ 18 19 20 21 ]. The assessment of infection severity was conducted in adherence to the Common Terminology Criteria for Adverse Events (CTCAE) version 5.0 [ 22 ]. All patients with grade 3 and 4 events were hospitalized and treated at our department. A positive blood culture indicating bacteremia was defined as the growth of organisms in one or more bottles, excluding coagulase-negative staphylococci, which required two separate positive blood cultures with matching antibiograms to confirm true bacteremia. Three blood cultures were performed, utilizing media for aerobic bacteria, anaerobic bacteria, and fungi. Sepsis was identified as an acute increase in Sequential Organ Failure Assessment score of 2 points or more under suspicion of infection. Infectious events at grade 2 were identified and diagnosed through physical examinations and laboratory tests conducted during scheduled post-AHSCT visits either at the Hematology Outpatient Clinic or at the patient's primary care physician's office if they visited there initially. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection testing involved reverse transcription and real-time PCR amplification, utilizing either the Allplex SARS-CoV-2 Assay CE-IVD kit (Seegene, Seoul, Korea) or an antigen test capable of detecting SARS-CoV-2, respiratory syncytial virus and influenza A/B (AllTest Biotech, Hangzhou, China).

2. Ethics statement

The research adhered to the guidelines of good clinical and laboratory practices as well as the principles outlined in the Declaration of Helsinki. Approval for all procedures was obtained from the local ethical committee (The Ethical Committee of the Medical University of Lodz, Protocol No RNN/424/19/KE). Prior to participation, each patient provided informed consent for all examinations and procedures.

3. Sample collection

Blood samples were collected from all patients at the same time point- on day +14 after transplantation. At that time, most of the patients had already achieved engraftment and none of the patients had been discharged from the hospital before that time. At 4°C, serum was separated by centrifugation at 1,500 g for fifteen minutes. Until examination, the serum samples were stored at -80°C.

4. RT-qPCR

Total RNA, including miRNA, was extracted from 200 µL volume of serum using the miRNeasy Serum/Plasma Advanced Kit (QIAGEN, Hilden, Germany), following a phenol-free protocol, in accordance with the manufacturer’s instructions. Total RNA was eluted from the column using 20 µL of RNase-free water and stored at −20°C until further use.

The levels of specific miRNAs were measured through RT-qPCR using the miRCURY LNA miRNA Custom PCR Panels from QIAGEN. In summary, 2 µL of cDNA was generated from the total RNA, inclusive of mature miRNAs (<200 bp), employing the miRCURY LNA Reverse Transcription Kit (QIAGEN), following the manufacturer's instructions. The reaction took place at 42°C for 60 min, followed by inactivation at 95°C for 5 min using a TProfessional thermal cycler (Biometra, Analytik Jena, Jena, Germany). The cDNA was stored at −20°C until further analysis.

Subsequently, a mixture containing 3 µL of cDNA template (diluted 1:30), 5 µL of 2X miRCURY LNA SYBR Green PCR Master Mix, and supplemented with RNase-free water to reach a final volume of 10 µL, was dispensed into the PCR plate. Real-time PCR was conducted using a LightCycler480 II Real-Time PCR System (Roche, Pleasanton, CA, USA). The reaction commenced with an initial denaturation step at 95°C for 2 minutes, followed by 55 cycles of amplification at 95°C for 10 seconds and 56°C for 1 minute. Absolute quantification of miRNA was determined utilizing the LightCycler ® 480 Software , (Version 1.5, Roche, Mannheim, Germany).

5. Statistical analysis

MiRNA expression levels were determined employing the ΔCt method. RT-qPCR data normalization was conducted by utilizing the average expression levels of two miRNAs within each sample (hsa-miR-27b-3p and hsa-miR-148b-3p), identified as the most stable factors through analysis with the NormiRazor Software in a prior investigation involving AHSCT recipients [ 23 24 25 ]. The formula for calculating the normalized Ct values was: Normalized ΔCt=(mean Ct of hsa-miR-27b-3p and hsa-miR-148b-3p) − Ct miRNA of interest. This approach results in higher values for higher miRNA expression.

Differential expression analysis was carried out utilizing an unequal variances t -test. Group differences in continuous variables were assessed using Student’s t -test. Logistic univariate and multivariate regression analyses were performed to determine the association between specific miRNAs, clinical variables, and the incidence of infectious adverse events within the first 100 days following AHSCT. Final logistic regression model was validated using 10-fold cross-validation method.

All statistical analyses were carried out utilizing Statistica Version 13.1 (TIBCO, Palo Alto, CA, USA) and the R programming language (version 4.0.2). P -values less than 0.05 were regarded as statistically significant. Normalized miRNA expression data, along with key variables, were included in Supplementary File 1 . The target genes of analyzed miRNAs were predicted using miRNet 2.0 ( http://www.mirnet.ca/ , Accessed 10 October 2023) [ 26 ]. Using the miRNet, an enrichment analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways was conducted to investigate the role of selected miRNAs in biological processes.

Results

1. Characteristics of a study group

The study group consisted of 58 patients and the median age was 59 years (interquartile range [IQR], 49–65). Detailed characteristics of the study cohort are presented in Table 1 . Among the patients, 27 (46.6%) were males and 31 females (53.4%). The primary diagnoses of the patients included MM for 43 individuals (74.1%) and lymphoma for 15 individuals (25.9%). Among MM patients, 14 individuals (32.6%) were categorized as ISS I, 13 (30.2%) as ISS II, 16 (37.2%) as ISS III. Among lymphoma patients, 1 (6.7%) was classified as stage I, 2 (13.3%) stage II, 4 (26.7%) stage III, and 7 (46.7%) stage IV. A total of 23 patients (39.7%) had previously undergone two or more lines of therapy before AHSCT. Furthermore, 11 lymphoma patients (73.3%) received the BeEAM conditioning regimen, while 4 (26.7%) received the BEAM regimen. 29 MM patients (67.4%) were treated with melphalan-200, and 14 (32.6%) with reduced melphalan. During the AHSCT, 23 patients (39.7%) experienced infectious complications before discharge. Regarding clinical parameters at the discharge, 7 (12.1%) had elevated creatinine levels, and 29 (50%) had h a emoglobin levels less than or equal to 10 g/dL.

Fifteen patients (25.9%) experienced infectious complications after discharge within the initial +100 days post-transplantation. The median time to infection onset was 44 days (IQR, 25–78). The assessment of these infections resulted in the classification of 11 cases (73.3%) as Grade 2, and 3 (20.0%) as Grade 3 according to the CTCAE. Most infectious events diagnosed and treated in the outpatient clinic were lower and upper respiratory tract infections; one patient had confirmed SARS-CoV-2 infection using RT-PCR on nasopharyngeal swab specimen.

Four patients (26.7%) required hospitalization due to severe infection. Among them, one patient had sepsis caused by Acinetobacter junii , the other had acute kidney injury caused by pyelonephritis with Escherichia coli confirmed in urine culture, and the other had radiologically confirmed bilateral pneumonia. One patient (6.7%) experienced septic shock requiring vasopressor agents to correct hypotension with subsequent diagnosis of recurrent Clostridioides difficile infection. Further detailed information regarding infectious events can be found in Table 2 .

2. Factors affecting infectious complications occurrence

Overexpression of hsa-miR-361-3p (fold change [FC], 1.79; P =0.0139) in the patients experiencing infectious complications and overexpression of hsa-miR-320c (FC, 2.14; P <0.0001) in patients requiring hospitalization were observed ( Table 3 , Fig. 1 ). In the univariate logistic regression analysis, hsa-miR-361-3p expression (odds ratio [OR], 2.36; 95% confidence interval [CI], 1.21–4.62; P =0.0121) significantly influenced infection occurrence. Among clinical variables, only lymphoma diagnosis compared to MM diagnosis was related to higher occurrence of post-discharge infectious complications in univariate analysis (OR, 3.83; 95% CI, 1.07–13.66; P =0.0387) ( Table 4 ). In the multivariate model, both lymphoma diagnosis (OR, 6.88; 95% CI, 1.55–30.56; P =0.0112) and high expression of hsa-miR-361-3p (OR, 3.00; 95% CI, 1.40–6.41; P =0.0047) were independent factors associated with post-discharge infectious complications occurrence ( Table 4 ). Our model in 10-fold cross-validation preserved its diagnostic potential with an area under the receiver operating characteristics curve-of 0.78 (95% CI, 0.64–0.92) and at optimal cut-off reached 73.3% sensitivity and 79.1% specificity ( Fig. 2 ).

3. Prediction of miRNAs target genes and KEGG pathway analysis

In the next step, the target genes of two significant miRNAs- hsa-miR-361-3p and hsa-miR-320c were predicted by the miRNet using miRTarBase v8.0 database. Overall, these two miRNAs targeted a total of 250 genes- hsa-miR-361-3p- 144 genes, hsa-miR-320c- 108 genes. Two genes were targeted by both investigated miRNAs. In the next step, functional enrichment analysis of identified targets was performed using hypergeometric testing with the KEGG database ( Fig. 3 ). Overall, KEGG pathway analysis demonstrated that miRNAs deregulated in AHSCT recipients experiencing post-discharge infectious events were enriched for genes associated with several processes related to antimicrobial response, including transforming growth factor (TGF)-beta signaling pathway, Wnt signaling pathway, Herpes simplex infection and T-cell receptor signaling pathway.

Discussion

In this study, we explored selected serum miRNAs expression- hsa-miR-155-5p, hsa-miR-320c, and hsa-miR-361-3p in ASHSCT recipients and its association with post-transplant discharge early infection occurrence. We found that patients who experienced infectious adverse events and required hospitalization had higher serum expression of hsa-miR-361-3p and has-miR-320c, respectively. Our study is one of the few studies assessing serum miRNAs with AHSCT complications. Contrary to previous studies, which mainly focused on AHSCT outcomes, often only in MM patients, we showed that miRNAs are potentially useful for prediction of procedure-related infectious complications [ 24 25 27 28 29 ].

The miRNAs chosen for analysis in our study are established biomarkers for infectious diseases [ 30 ]. For example, Mice deficient in miR-155 exhibit weakened immune responses when faced with infections caused by Citrobacter rodentium , Salmonella typhimurium , and Listeria monocytogenes [ 31 32 33 ]. Examination of the transcriptome in CD4+ T cells lacking bic/microRNA-155 revealed a diverse array of genes under the regulation of microRNA-155. These genes encompass variety of molecular processes, such as T cell costimulation ( e.g. , TNFSF9 ), chemotaxis ( e.g. , CCL5 ), and signaling ( e.g. , IKBKE ) [ 32 ]. Moreover, miR-155 levels have been observed to rise in peripheral monocytes and in the serum of patients with chronic Hepatitis C virus (HCV) infection and in murine bone marrow-derived macrophages subjected to stimulation with lipopolysaccharide (LPS) and interferon-γ [ 34 35 ].

Similarly, elevated expression levels of circulating serum miR-320c, together with miR-134, and miR-483-5p were observed in HCV-infected patients compared to healthy controls [ 36 ]. Recently, miR-320c was reported to be upregulated in patients with coronavirus disease 2019, and higher expression of this miRNA correlated with the severity of the disease. In our study, we demonstrated a significant upregulation of circulating miR-320c in patients with severe infections (grade 3 and 4) necessitating hospitalization. Regrettably, a limited number of hospitalized patients (n=4) precludes the establishment of a statistical model for utilizing this miRNA in predicting the most severe infections. Nevertheless, this intriguing observation warrants further investigation in future studies involving larger populations.

Several reports connect the overexpression of miR-320c with respiratory tract infections and pulmonary diseases. Analysis of miR-320c expression in patient blood samples revealed significantly increased expression of miR-320c in individuals with alpha-1 antitrypsin (AAT) deficiency [ 37 ]. In addition, miR-320c exhibited increased expression in individuals with obstructive lung diseases (including bronchiectasis, emphysema, chronic bronchitis, and asthma) compared to healthy individuals, irrespective of their AAT status. In the same study, authors found that HL60 cells treated with the pro-inflammatory factor LPS showed increase in miR-320c expression, suggesting that miR-320c responds to inflammation. In another study, mir-320c was found to be significantly upregulated, together with miR-200c-3p, and miR-449c-5p in bronchoalveolar lavage in patients with chronic obstructive pulmonary disease (COPD), chronic inflammatory disease commonly induced by cigarette smoke [ 38 ]. Similarly, hsa-miR-320c was upregulated in the blood plasma of patients COPD and asthma-COPD overlap syndrome [ 39 ].

We also conducted functional network analysis of miRNAs to determine their connections with particular pathways and revealed that both miRNAs, hsa-miR-361-3p and hsa-miR-320c, regulate expression of genes involved in TGF-β signaling pathway and Wnt signaling pathway. All immune cell types, including B-cells, T-cells, dendritic cells, and macrophages, release TGF-β, inhibiting their proliferation, differentiation, and response to other cytokines. Consequently, TGF-β acts as a strong immunosuppressor, with disruptions in its signaling associated with autoimmunity and inflammation [ 40 ]. For example, elevated levels of circulating TGF-β1 are associated with increased severity and mortality in severe community-acquired pneumonia and acute respiratory distress syndrome induced by sepsis [ 41 42 ]. Especially, we found that latent TGF-β activator- Thrombospondin-1 (THBS-1) may be targeted by miR-320c. It was reported that THBS-1 improves neutrophil motility and respiratory burst promotes M1 macrophage recruitment and cytotoxic prosperities [ 43 44 ]. In our previous study, we demonstrated that lymphoma patients who developed bacteremia during the AHSCT procedure had lower THBS-1 concentration compared with those without. In addition, patients with lower THBS-1 concentrations experienced febrile neutropenia earlier than those without [ 45 ]. All this strongly supports our hypothesis that miR-361-3p and miR-320c are novel, major regulators of crucial immune processes whose activities translate into the risk of early infection after post-transplant discharge in AHSCT recipients.

Recently, transcriptomics has been applied to predict bacteremia in cancer patients. For example, Labiad et al. used high-throughput transcriptomic analysis of peripheral blood mononuclear cells (PBMCs) to identify an 11-gene signature predicting systemic inflammatory response syndrome or sepsis post-AHSCT [ 46 ]. Their classification model, constructed from samples collected just after the conditioning regimen, showed early predictive capability. In our study, contrary to the above study protocol, we focused on complications that occur after post-transplant discharge.

The emphasis on serum miRNAs in our study is driven by their clinical accessibility, reliability, and ease of integrating blood-based biomarker tests into clinical applications. Specifically in the context of AHSCT, serum miRNAs may have the capacity to capture systemic changes, reflecting the overall response of the entire organism to the AHSCT procedure. In contrast, PBMCs and their cytokine products constitute a specific subset of cells directly influenced by the procedure. Serum miRNAs, released into circulation by various cell types, encompassing both hematopoietic and non-hematopoietic cells, offer a more comprehensive insight into the early onset of complications [ 47 ].

The limitations of our study lie in its single-center character. Nevertheless, as with any biomarker studies, validating our results in an independent dataset, preferably recruited in a different center, would significantly strengthen their clinical relevance.

Identifying specific complications-associated miRNAs can guide risk stratification and inform treatment decisions, such as selecting preventative or supportive care interventions. The timely detection of individuals at risk of developing infectious complications can optimize the prophylactic use of antimicrobial agents tailored to the specific risk profile of each individual. Distinguishing between patients with high and low infectious risk following AHSCT can facilitate the selection of patients with a safe toxicity profile. It could enable early hospital discharge or outpatient AHSCT, promoting a more balanced allocation of hospital resources while ensuring optimal safety. However, this should be evaluated in a prospective clinical trial.

In conclusion, our study established that high hsa-miR-361-3p serum expression is an independent risk factor for infectious complications after post-transplant discharge in AHSCT recipients. Our study demonstrates serum miRNAs' clinical relevance and prospective utility as biomarkers for enhancing patient outcomes during AHSCT.

Funding: The study was funded by the National Science Center grant number 2019/33/B/NZ5/00536 awarded to WF and 2022/45/N/NZ6/02904 awarded to DM.

Conflict of Interest: AW: Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Astellas: Consultancy, Honoraria; Celgene/BMS: Honoraria; JazzPharmaceuticals/swixx: Honoraria; Novartis: Honoraria; Gilead: Honoraria; Pfizer: Honoraria; Servier: Honoraria. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Author Contributions: Conceptualization: DM, WF. Data curation: DM, KK, MN, MM, PS. Formal analysis: DM, KK, WF. Funding acquisition: DM, WF. Investigation: DM, ID. Methodology: DM, ID, EP. Project administration: DM. Resources: DM, MN, MM, PS, AW, WF. Software: DM, KK. Supervision: AW, WF. Validation: DM, EP, AW, WF. Visualization: DM, KK. Writing - original draft: DM. Writing - review & editing: DM, EP, AW, WF.

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SUPPLEMENTARY MATERIAL

Supplementary File 1

Normalized miRNA expression data with clinical variables- infection and hospitalization occurrence.