The timely reconstitution and regain of function of a donor-derived immune system is of utmost importance for the recovery and long-term survival of patients after allogeneic hematopoietic stem cell ...transplantation (HSCT). Of note, new developments such as umbilical cord blood or haploidentical grafts were associated with prolonged immunodeficiency due to delayed immune reconstitution, raising the need for better understanding and enhancing the process of immune reconstitution and finding strategies to further optimize these transplant procedures. Immune reconstitution post-HSCT occurs in several phases, innate immunity being the first to regain function. The slow T cell reconstitution is regarded as primarily responsible for deleterious infections with latent viruses or fungi, occurrence of graft-versus-host disease, and relapse. Here we aim to summarize the major steps of the adaptive immune reconstitution and will discuss the importance of immune balance in patients after HSCT.
Since the early beginnings, in the 1950s, hematopoietic stem cell transplantation (HSCT) has become an established curative treatment for an increasing number of patients with life-threatening ...hematological, oncological, hereditary, and immunological diseases. This has become possible due to worldwide efforts of preclinical and clinical research focusing on issues of transplant immunology, reduction of transplant-associated morbidity, and mortality and efficient malignant disease eradication. The latter has been accomplished by potent graft-versus-leukemia (GvL) effector cells contained in the stem cell graft. Exciting insights into the genetics of the human leukocyte antigen (HLA) system allowed improved donor selection, including HLA-identical related and unrelated donors. Besides bone marrow, other stem cell sources like granulocyte-colony stimulating-mobilized peripheral blood stem cells and cord blood stem cells have been established in clinical routine. Use of reduced-intensity or non-myeloablative conditioning regimens has been associated with a marked reduction of non-hematological toxicities and eventually, non-relapse mortality allowing older patients and individuals with comorbidities to undergo allogeneic HSCT and to benefit from GvL or antitumor effects. Whereas in the early years, malignant disease eradication by high-dose chemotherapy or radiotherapy was the ultimate goal; nowadays, allogeneic HSCT has been recognized as cellular immunotherapy relying prominently on immune mechanisms and to a lesser extent on non-specific direct cellular toxicity. This chapter will summarize the key milestones of HSCT and introduce current developments.
Acute graft-versus-host disease (aGvHD) is a major cause of adverse outcome in hematopoietic stem cell transplantation (HSCT), with a high incidence (20-50%). A novel, non-invasive diagnostic test to ...predict for prevalence and severity would enable improved prophylaxis and reduce morbidity. Circulatory microRNAs (miRNAs) miR-423, miR-199, miR-93*, and miR-377 have previously been associated with aGvHD in post-HSCT patient plasma, but validation is lacking and their expression within extracellular vesicles (EVs) has not been explored. This study replicated elevated serum expression of miR-423 (
< 0.001), miR-199 (
= 0.04), miR-93* (
< 0.001), and miR-377 (
= 0.03) in aGvHD, using a prognostic cohort of day 14 (D14) post-HSCT patient samples (
= 81). Expression also associated with disease severity. Further analysis at aGvHD diagnosis in an independent cohort (
= 65) confirmed high miR-423 (
= 0.02), miR-199 (
= 0.007), and miR-93* (
= 0.004) expression at disease onset. Investigation of expression patterns during early HSCT sequential timepoints (pre-HSCT to D28) identified elevated miRNAs at D7 post-HSCT in all transplant patients. In a novel investigation of miRNA expression in serum EVs (
= 15), miR-423 (
= 0.09), miR-199 (
= 0.008), and miR-93* (
= 0.001) levels were lower at D14 in patients who later developed aGvHD, and this was replicated for miR-423 (
= 0.02) and miR-199 (
= 0.04) (
= 47). Comparing serum to circulating EVs, at D14 patients remaining aGvHD-free had higher expression of miR-423 (
= 0.03), miR-199 (
= 0.009), and miR-93* (
= 0.002) in the EV fraction. Results verify the capacity for circulating miR-423, miR-199, and miR-93* as diagnostic and prognostic aGvHD biomarkers. The novel finding of their differential expression in EVs suggests a potential role in aGvHD etiology.
Acute graft vs. host disease (aGvHD) is a frequent complication following allogeneic haematopoeitic transplantation (HSCT). Despite recent advances, there are no universally accepted biomarkers to ...determine development of aGvHD. MicroRNAs miR-146a and miR-155 have been previously associated with aGvHD and show promise as clinically translatable biomarkers. In this study, we performed comprehensive expression profiling of miR-146a, miR-155, and miR-155
expression in aGvHD target tissue and biofluids and relate expression to post-HSCT outcomes.
MicroRNA expression was assessed by qRT-PCR in gastrointestinal (
= 31) and skin (
= 31) biopsies as well as serum (exploratory cohort
= 34, verification cohort
= 81, diagnostic cohort
= 65) and urine (exploratory cohort
= 30, verification cohort
= 56, diagnostic cohort
= 20) biofluids, including extracellular vesicle (EV) cohorts (serum EV
= 15, urine EV
= 30). Expression was related to aGvHD incidence, severity and overall survival.
In GI samples, expression of miR-155 (
= 0.03) and miR-146a (
= 0.03) was higher at aGvHD onset compared to patients with no GvHD. In skin biopsies, expression of miR-155 (
= 0.004) was upregulated in aGvHD patients compared to normal control skin. Expression of miR-146a was higher in aGvHD compared to no aGvHD biopsies (
= 0.002). In serum, miR-155 (
= 0.03) and miR-146a (
= 0.02) expression was higher at day 14 (D14), while in urine expression was elevated at D7 post-HSCT in patients who developed aGvHD compared to those disease-free. This was verified in an independent serum (miR-155
= 0.005, miR-146a
= 0.003) and urine (miR-155
= 0.02, miR-146a
= 0.04) cohort, where both microRNAs were also associated with aGvHD by ROC analysis. In serum and urine samples taken at the time of aGvHD symptoms, expression of miR-155 and miR-146a was also elevated (serum miR-155
= 0.03, miR-146a
< 0.001; urine miR-155
= 0.02, miR-146a
= 0.02). In contrast, miR-146a and miR-155 were downregulated at D14 in serum EVs and at D7 in urine EVs in patients who developed aGvHD compared to those that remained disease-free, in both an exploratory (serum miR-155
= 0.02, miR-146a
= 0.06; urine miR-155
= 0.02, miR-146a
= 0.07) and an independent cohort (serum miR-155
= 0.01, miR-146a
= 0.02).
These results further support a role for miR-155 and miR-146a as non-invasive, clinically relevant biomarkers for aGvHD. However, the link between their involvement in generalized inflammation and in specific pathophysiology requires further investigation at a systemic level.
Acute graft-versus-host disease (aGvHD) is the most frequent and serious complication following hematopoietic stem cell transplantation (HSCT), with a high mortality rate. A clearer understanding of ...the molecular pathogenesis may allow for improved therapeutic options or guide personalized prophylactic protocols. Circulating microRNAs are expressed in body fluids and have recently been associated with the etiology of aGvHD, but global expression profiling in a HSCT setting is lacking. This study profiled expression of
= 799 mature microRNAs in patient serum, using the NanoString platform, to identify microRNAs that showed altered expression at aGvHD diagnosis. Selected microRNAs (
= 10) were replicated in independent cohorts of serum samples taken at aGvHD diagnosis (
= 42) and prior to disease onset (day 14 post-HSCT,
= 47) to assess their prognostic potential. Sera from patients without aGvHD were used as controls. Differential microRNAs were investigated
for predicted networks and mRNA targets. Expression analysis identified 61 microRNAs that were differentially expressed at aGvHD diagnosis. miR-146a (
= 0.03), miR-30b-5p (
= 0.007), miR-374-5p (
= 0.02), miR-181a (
= 0.03), miR-20a (
= 0.03), and miR-15a (
= 0.03) were significantly verified in an independent cohort (
= 42). miR-146a (
= 0.01), miR-20a (
= 0.03), miR-18 (
= 0.03), miR-19a (
= 0.03), miR-19b (
= 0.01), and miR-451 (
= 0.01) were differentially expressed 14 days post-HSCT in patients who later developed aGvHD (
= 47). High miR-19b expression was associated with improved overall survival (OS) (
= 0.008), whereas high miR-20a and miR-30b-5p were associated with lower rates of non-relapse mortality (
= 0.05 and
= 0.008) and improved OS (
= 0.016 and
= 0.021). Pathway analysis associated the candidate microRNAs with hematological and inflammatory disease. Circulating biofluid microRNAs show altered expression at aGvHD onset and have the capacity to act as prognostic and diagnostic biomarkers. Their differential expression in serum suggests a role for circulatory microRNAs in aGvHD pathology, which warrants further investigation.
Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the main curative therapy for hematological malignancy such as leukemias, lymphomas, or multiple myelomas and some other ...hematological disorders. In this therapy, cure of hematological diseases relies on graft-versus-malignancy effects by allogenic immune cells. However, severe posttransplant treatment-associated complications such as acute graft-versus-host disease (aGvHD) and chronic graft-versus-host disease (cGvHD) limit this approach. Most research into GvHD has concentrated on the aGvHD, while the more complex and multifaceted chronic form has been largely poorly investigated. cGvHD is a multi-organ autoimmune disorder and is the major cause of non-relapse morbidity and mortality following allo-HSCT, occurring in about 50% of patients, or 13,000-15,000 patients per year worldwide. Therefore, there is a high medical need for an early prediction of these therapy-associated toxicities. Biomarkers have gained importance over the last decade in diagnosis, in prognosis, and in prediction of pending diseases or side effects. Biomarkers can be cells, factors isolated from target tissues, or soluble factors that can be detected in body fluids. In this review, we aim to summarize some of the recent developments of biomarkers in the field of allo-HSCT. We will focus on cell-based biomarkers (B-cell subsets) for cGvHD and soluble factors including microRNA (miRNA), which are excreted into serum/plasma and urine. We also discuss the potential role of cytosolic and extracellular 70 kDa heat shock proteins (HSP70) as potential biomarkers for aGvHD and their role in preclinical models. Proteomic biomarkers in the blood have been used as predictors of treatment responses in patients with aGvHD for many years. More recently, miRNAs have been found to serve as a biomarker to diagnose aGvHD in the plasma. Another development relates to urine-based biomarkers that are usually detected by capillary electrophoresis and mass spectrometry. These biomarkers have the potential to predict the development of severe aGvHD (grades III-IV), overall mortality, and the pending development of cGvHD in patients posttransplant.
The reconstitution and the regaining of function of a healthy, transplanted immune system is of utmost importance for the recovery and long term survival of patients after HSCT. New developments ...within HSCT, for example, umbilical cord blood or haploidentical grafts, both leading to prolonged immunodeficiency and delayed immune reconstitution, have increased the need to improve immune reconstitution. Thus, understanding and enhancing immune reconstitution post-HSCT is an area of intense research. Immune reconstitution post-HSCT occurs in several steps, innate immunity being the first to regain function. Although the slow T-cell reconstitution is regarded as primarily responsible for deleterious infections with latent viruses or fungi, occurrence of graft-versus-host disease, and relapse, the importance of innate immune cells for disease and infection control is being reevaluated. Here we aim to summarize the major steps of the immune reconstitution in patients after HSCT.
Introduction
MicroRNAs are small, non-coding single-stranded RNAs and regulate approximately 50% of all genes by repressing translation. They are present in bodily fluids, where they are protected ...from RNase-mediated degradation by encapsulation into extracellular vesicles (EVs) and demonstrate a novel capacity to regulate the cellular differentiation of blood cells and immune function. Candidate microRNAs miR-377, miR-199, miR-93* and miR-423 have previously been associated with acute graft versus host disease (aGvHD) in post-hematopoietic stem cell transplant (HSCT) patient plasma. However, validation in independent cohorts is necessary, as well as further exploration to assess expression in the EV fraction of the blood.
Methods
MicroRNA expression was evaluated in early HSCT time point exploratory (n=34), validation (n=47) and diagnostic (n=65) serum cohorts by TaqMan qRT-PCR. Expression was also assessed in serum EVs (exploratory n=16 and validation n=47 cohorts) by EV isolation, RNA extraction and TaqMan qRT-PCR analysis.
Results
In sequential pre- and post-HSCT serum samples (n=34; pre-HSCT, Day0 (D0), D7, D14 & D28), miR-423 (p=0.03), miR-199 (p=0.06), miR-93* (p=0.04) and miR-377 (p=0.03) were upregulated at D14 in patients who developed aGvHD vs. no aGvHD. MiR-423 was also significantly upregulated at D0 (p=0.04), D7 (p=0.03) and D28 (p=0.03) in aGvHD patients. In relation to aGvHD severity, miR-423 (p=0.05), miR-199 (p=0.007) and miR-93* (p=0.09) were differentially expressed at D14 according to aGvHD grade. MiR-423 (p=0.02), miR-199 (p=0.07) and miR-93* (p=0.01) expression was validated at D14 in an independent cohort (n=47).
When the exploratory and validation D14 samples were combined (n=81), miR-423 (p<0.001), miR-199 (p=0.04) and miR-93* (p<0.001) expression was upregulated in patients that developed aGvHD vs. no GvHD and ROC analysis identified miR-423 (p<0.001, AUC=0.75), miR-199 (p=0.09, AUC=0.62) and miR-93* (p<0.001, AUC=0.74) to have diagnostic ability. MiR-423 (p=0.001), miR-199 (p=0.01) and miR-93* (p<0.001) expression was higher in severe (III-IV) vs. no aGvHD, miR-423 (p=0.006) and miR-93* (p=0.01) were higher in mild (I-II) vs. no aGvHD and miR-199 was higher in severe (III-IV) vs. mild (I-II) aGvHD (p=0.002). All microRNAs demonstrated significant positive correlation (p<0.001), thus, principle component analysis (PCA) was performed. The PC1 composite score was used for ROC analysis and showed diagnostic ability for aGvHD incidence (p<0.001, AUC=0.73).
In an independent diagnostic cohort (n=65), from a separate Institution, miR-423 (p=0.02), miR-199 (p=0.007) and miR-93* (p=0.004) expression was higher at aGvHD onset and showed diagnostic ability by ROC analysis (miR-423 p=0.03 AUC=0.66; miR-199 p=0.04 AUC=0.65; and miR-93* p=0.01 AUC=0.68). The three microRNAs had diagnostic ability with respect to aGvHD incidence based on composite ROC analysis of PC1 (p=0.019, AUC=0.68).
MicroRNAs were also investigated within serum EVs (n=15). MiR-199 (p=0.008), miR-93* (p=0.001) and miR-423 (p=0.09) expression was lower at D14 in patients who developed aGvHD. Results were confirmed in a D14 validation cohort (n=47), with lower EV miR-423 (p=0.02) and miR-199 (p=0.04), but not miR-93* (p=0.15) expression in patients who developed aGvHD. MiR-423 and miR-199 had diagnostic ability based on composite PC1 ROC analysis (p=0.06, AUC=0.69). By D14, patients remaining aGvHD free had higher expression of miR-423 (p=0.03), miR-199 (p=0.05) and miR-93* (p<0.001) in the EV fraction compared to whole serum.
Conclusions
Results validate the capacity for circulating serum miR-423, miR-199 and miR-93* to act as diagnostic and prognostic biomarkers for aGvHD. Novel findings of differential expression between whole serum and the EV compartment prior to disease onset suggest a role for EV microRNAs in the biology of aGvHD, which warrants further investigation.
No relevant conflicts of interest to declare.
Introduction
MicroRNAs are expressed in body fluids and have recently been associated with the etiology of acute graft versus host disease (GvHD), but global expression profiling in a haematopoietic ...stem cell transplant (HSCT) setting is lacking. An improved understanding of the molecular pathogenesis of aGvHD may allow for improved therapeutic options, or guide personalised prophylactic protocols.
Methods
Mature microRNA serum expression (n=799) was assessed using nCounter technology (NanoString) in a training cohort of diagnostic aGvHD samples (n=12). Analysis assessed for microRNAs that were dysregulated in patients who developed aGvHD compared to no aGvHD. Signature microRNAs were validated in an independent cohort of serum samples taken at aGvHD diagnosis (n=42) and prior to disease onset (day 14 (D14) post-HSCT, n=47).
Results
NanoString profiling identified 61 microRNAs that were differentially expressed at aGvHD onset, of which n=27 were downregulated (fold change (FC) -6.94 - -1.75; p<0.01 - p=0.048) in aGvHD while n=34 were upregulated (FC 1.35 - 5.41; p<0.01 - p=0.046). 10 microRNAs (miR-146a, miR-30b, miR-374, miR-20a, miR-15a, miR-181a, miR-18a, miR-19a, miR-19b and miR-451a) were selected for further assessment in an independent diagnostic cohort (n=42), based on high FC or those reported in the literature to be implicated in GvHD, T-cell function or the inflammatory response. MiR-146a (p=0.03), miR-30b-5p (p=0.007), miR-374-5p (p=0.02) and miR-181a (p=0.03) were significantly downregulated, whilst miR-20a (p=0.03) and miR-15a (p=0.03) were significantly upregulated in aGvHD. MiR-30b (AUC=0.75, p=0.007), miR-374-5p (AUC=0.74, p=0.01) and miR-15a (AUC=0.70, p=0.04) had diagnostic ability for aGvHD as assessed by receiver operator characteristic (ROC) analysis, whilst miR-181 (AUC=0.68, p=0.06), miR-146a (AUC=0.66, p=0.09) and miR-20a (AUC=0.68, p=0.06) were approaching significance. There was no significant difference in microRNA expression between clinical factors including transplant relationship, patient gender or conditioning regimen, indicating the effect to be related to aGvHD incidence.
MicroRNA expression was also assessed at D14 post-HSCT in an independent cohort (n=47) to investigate their prognostic marker potential. MiR-146a (p=0.01), miR-20a (p=0.03), miR-18a (p=0.03), miR-19a (p=0.03), miR-19b (p=0.02) and miR-451 (p=0.01) were expressed at significantly higher levels in patients who developed aGvHD vs. no aGvHD. In ROC analysis, miR-146a (A=0.68, p=0.03), miR-19b (A=0.70, p=0.02) and miR-451 (A=0.69, p=0.03) had diagnostic ability with regards to aGvHD incidence, whilst miR-18a (A=0.65, p=0.09), miR-19a (A=0.65, p=0.08) and miR-20a (A=0.67, p=0.06) were approaching significance.
Conclusions
Circulating microRNAs have the capacity to act as prognostic and diagnostic biomarkers for aGvHD and might assist in developing personalised therapeutic approaches. Their dysregulated expression suggests a role in aGvHD pathology, which warrants further investigation.
No relevant conflicts of interest to declare.
Abstract Chronic graft-versus-host disease (cGVHD) is a serious and frequent complication of allogeneic hematopoietic stem cell transplantation (HCT). Currently, no biomarkers for prediction and ...diagnosis of cGVHD are available. We performed a large prospective study focusing on noninvasive biomarkers for National Institutes of Health–defined cGVHD patients (n = 163) in comparison to time-matched HCT recipients who never experienced cGVHD (n = 64), analyzed from day 100 after HCT. In logistic regression analysis, CD19+ CD21low B cells ( P = .002; hazard ratio HR, 3.31; 95% confidence interval CI, 1.53 to 7.17) and CD4+ CD45RA+ CD31+ T cells ( P < .001; HR, 3.88; 95% CI, 1.88 to 7.99) assessed on day 100 after HCT were significantly associated with subsequent development of cGVHD, independent of clinical parameters. A significant association with diagnosis of cGVHD was only observed for CD19+ CD21low B cells ( P = .008; HR, 3.00; 95% CI, 1.33 to 6.75) and CD4+ CD45RA+ CD31+ T cells ( P = .017; HR, 2.80; 95% CI, 1.19 to 6.55). CD19+ CD21low B cells were found to have the highest discriminatory value with an area under the receiver operating curve of .77 (95% CI, .64 to .90). Our results demonstrate that CD19+ CD21low B cells and CD4+ CD45RA+ CD31+ T cells are significantly elevated in patients with newly diagnosed cGVHD.
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