Optimizing anti-T-lymphocyte globulin dosing to improve long-term outcome after unrelated hematopoietic cell transplantation for hematologic malignancies.

Prophylaxis of graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (HCT) remains challenging. Because prospective randomized trials of in-vivo T cell depletion using anti-T-lymphocyte globulin (ATLG) in addition to a calcineurin inhibitor and methotrexate (MTX) led to conflicting outcome results, we evaluated the impact of ATLG on clinical outcome, lymphocyte- and immune reconstitution survival models. In total, 1500 consecutive patients with hematologic malignancies received matched unrelated donor (MUD) HCT with cyclosporin and MTX (N = 723, 48%) or with additional ATLG (N = 777, 52%). In the ATLG cohort, grades III-IV acute (12% vs 23%) and extensive chronic GVHD (18% vs 34%) incidences were significantly reduced (P < .0001). Nonrelapse mortality (27% vs 45%) and relapse (30% vs 22%) differed also significantly. Event-free and overall survival estimates at 10 years were 44% and 51% with ATLG and 33% and 35% without ATLG (P < .002 and <.0001). A dose-dependent ATLG effect on lymphocyte- and neutrophil reconstitution was observed. At ATLG exposure, lymphocyte counts and survival associated through a logarithmically increasing function. In this survival model, the lymphocyte count optimum range at exposure was between 0.4 and 1.45/nL (P = .001). This study supports additional ATLG immune prophylaxis and is the first study to associate optimal lymphocyte counts with survival after MUD-HCT.

Ileostomy for steroid-resistant acute graft-versus-host disease of the gastrointestinal tract.

Steroid-resistant acute graft-versus-host disease (GVHD) of the gastrointestinal tract associates with important morbidity and mortality. While high-dose steroids are the established first-line therapy in GVHD, no second-line therapy is generally accepted. In this analysis of 65 consecutive patients with severe, steroid-resistant, intestinal GVHD (92% stage 4), additional ileostomy surgery significantly reduced overall mortality (hazard ratio 0.54; 95% confidence interval, 0.36-0.81; p = 0.003) compared to conventional GVHD therapy. Median overall survival was 16 months in the ileostomy cohort compared to 4 months in the conventional therapy cohort. In the ileostomy cohort, both infectious- and GVHD-associated mortality were reduced (40% versus 77%). Significantly declined fecal volumes (p = 0.001) after surgery provide evidence of intestinal adaptation following ileostomy. Correlative studies indicated ileostomy-induced immune-modulation with a > 50% decrease of activated T cells (p = 0.04) and an increase in regulatory T cells. The observed alterations of the patients' gut microbiota may also contribute to ileostomy's therapeutic effect. These data show that ileostomy induced significant clinical responses in patients with steroid-resistant GVHD along with a reduction of pro-inflammatory immune cells and changes of the intestinal microbiota. Ileostomy is a treatment option for steroid-resistant acute GVHD of the gastrointestinal tract that needs further validation in a prospective clinical trial.

Microbiologic Diagnostic Workup of Acute Respiratory Failure with Pulmonary Infiltrates after Allogeneic Hematopoietic Stem Cell Transplantation: Findings in the Era of Molecular- and Biomarker-Based Assays.

Allogeneic hematopoietic stem cell transplantation (HSCT) recipients frequently develop acute respiratory failure (ARF) with pulmonary infiltrates. Molecular- and biomarker-based assays enhance pathogen detection, but data on their yield in this population are scarce. This was a retrospective single-center study of 156 consecutive HSCT recipients admitted to the intensive care unit (ICU) between May 2013 and July 2017. Findings from a microbiologic diagnostic workup using currently available methods on bronchoalveolar lavage (BAL) and blood samples from 66 patients (age, 58 years [range, 45 to 64]; HSCT to ICU, 176 days [range, 85 to 407]) with ARF and pulmonary infiltrates were analyzed. In 47 patients (71%) a causative pathogen was identified (fungal, n = 28; viral, n = 26; bacterial, n = 18). Polymicrobial findings involving several pathogen groups occurred in 20 patients (30%). Culture (12/16, 75%), galactomannan (13/15, 87%), and Aspergillus-PCR (8/9, 89%) from BAL but not serum galactomannan (6/14, 43%) helped to diagnose invasive aspergillosis (n = 16, 24%). Aspergillus-PCR detected azole resistance in 2 cases. Mucorales was found in 7 patients (11%; BAL culture, n = 6; Mucorales-PCR, n = 1). Patients with identified pathogens had higher Simplified Acute Physiology Score II scores (P = .049) and inferior ICU survival (6% versus 37%, P < .01), which largely related to the presence of an invasive fungal infection. Eight patients (12%) had 1 or more viruses with uncertain lung pathogenicity as the sole microbiologic finding. A diagnostic microbiologic workup incorporating molecular- and biomarker-based assays identified pathogens in most HSCT recipients with ARF and pulmonary infiltrates admitted to the ICU. Implications of polymicrobial infection and pathogen patterns in these patients warrant further investigation.

High-dose melphalan-based sequential conditioning chemotherapy followed by allogeneic haematopoietic stem cell transplantation in adult patients with relapsed or refractory acute myeloid leukaemia.

Considering the unsatisfactory results of salvage therapies for patients with relapsed/refractory acute myeloid leukaemia (R/R-AML), their value before allogeneic haematopoietic stem cell transplantation (HSCT) remains questionable. However, direct allogeneic HSCT following established conditioning regimens applied in patients with R/R-AML during active disease has been equally disappointing. In this retrospective observational study, high-dose melphalan, as part of a sequential preparative regimen, followed by a total body irradiation (4 × 2 Gy)-based or a treosulfan-based dose-adapted conditioning therapy for allogeneic HSCT was administered to 292 adult patients (median age 56 years, range 17-74) with primary refractory (144 patients), secondary refractory (97 patients) or relapsed AML (51 patients). Overall survival rates at 3 years were 34%, 29% and 41%, respectively. Risk factors associated with an inferior survival were higher age, transplantation from a human leucocyte antigen-mismatched donor and high disease burden. Patients transplanted with blast infiltration <20% showed a notable survival rate of 51% at 3 years. In particular, patients with primary refractory AML showed a more favourable outcome when transplanted early during their disease course. Thus, high-dose melphalan-based sequential conditioning chemotherapy followed by an allogeneic HSCT is feasible and enables long-term remission to be achieved in a substantial proportion of patients with active R/R-AML.

Endoscopic and Histological Findings Are Predicted by Fecal Calprotectin in Acute Intestinal Graft-Versus-Host-Disease.

BACKGROUND: Gastrointestinal graft-versus-host-disease (GI-GVHD) is a major cause of nonrelapse mortality after hematopoietic stem cell transplantation (HSCT) necessitating endoscopic examinations and biopsies for diagnosis. Fecal calprotectin (CPT) has been widely used in gastrointestinal inflammation, but comprehensive data in GI-GVHD are lacking. AIMS: We aimed to identify an association of CPT with endoscopic findings, mucosal damage and symptoms for diagnosing and monitoring acute GI-GVHD. METHODS: Symptoms were prospectively evaluated in 110 consecutive HSCT recipients by standardized questionnaires and Bristol Stool Scale (BSS). CPT was assayed by ELISA. Symptom assessment and CPT were performed weekly and with onset of first symptoms. GVHD was diagnosed according to the Glucksberg criteria and by endoscopic biopsies. Patients with GI-GVHD received standard high-dose corticosteroid therapy and follow-up CPT, and symptom evaluation was performed after 28 days. Patients not responding to steroid treatment were re-evaluated by colonoscopy. RESULTS: GI-GVHD was diagnosed in 40 patients. Twelve patients with GI symptoms and CMV colitis and 24 patients with isolated skin GVHD were included as control subjects. CPT was significantly higher in GI-GVHD compared to skin GVHD and CMV colitis. Endoscopic findings, histological grading, abdominal cramps, diarrhea, urgency and BSS correlated with CPT. At follow-up, CPT correlated with abdominal cramps, diarrhea, urgency and BSS. In steroid refractory patients, CPT level was still significantly associated with severity of mucosal damage. CONCLUSION: CPT predicts endoscopic and histological findings in GI-GVHD and correlates with lower GI symptoms. It enables to discriminate GVHD from CMV colitis and to monitor therapeutic success.

Early human cytomegalovirus replication after transplantation is associated with a decreased relapse risk: evidence for a putative virus-versus-leukemia effect in acute myeloid leukemia patients.

The impact of early human cytomegalovirus (HCMV) replication on leukemic recurrence was evaluated in 266 consecutive adult (median age, 47 years; range, 18-73 years) acute myeloid leukemia patients, who underwent allogeneic stem cell transplantation (alloSCT) from 10 of 10 high-resolution human leukocyte Ag-identical unrelated (n = 148) or sibling (n = 118) donors. A total of 63% of patients (n = 167) were at risk for HCMV reactivation by patient and donor pretransplantation HCMV serostatus. In 77 patients, first HCMV replication as detected by pp65-antigenemia assay developed at a median of 46 days (range, 25-108 days) after alloSCT. Taking all relevant competing risk factors into account, the cumulative incidence of hematologic relapse at 10 years after alloSCT was 42% (95% confidence interval [CI], 35%-51%) in patients without opposed to 9% (95% CI, 4%-19%) in patients with early pp65-antigenemia (P < .0001). A substantial and independent reduction of the relapse risk associated with early HCMV replication was confirmed by multivariate analysis using time-dependent covariate functions for grades II to IV acute and chronic graft-versus-host disease, and pp65-antigenemia (hazard ratio = 0.2; 95% CI, 0.1-0.4, P < .0001). This is the first report that demonstrates an independent and substantial reduction of the leukemic relapse risk after early replicative HCMV infection in a homogeneous population of adult acute myeloid leukemia patients.

Long-Term Follow-Up after Adoptive Transfer of BK-Virus-Specific T Cells in Hematopoietic Stem Cell Transplant Recipients.

The BK virus (BKV) causes severe hemorrhagic cystitis in hematopoietic stem cell transplant (HSCT) recipients. To eliminate reactivated BKV, symptomatic patients can be treated with a reduction of the immunosuppressive therapy, with the antiviral drug cidofovir, or with virus-specific T cells (VSTs). In the current study, we compared the effect of VSTs to other treatment options, following up specific T cells using interferon-gamma ELISpot assay. We observed BKV large T-specific cellular responses in 12 out of 17 HSCT recipients with BKV-related cystitis (71%). In recipients treated with VSTs, 6 out of 7 showed specific T-cell responses, and that number in those without VSTs was 6 out of 10. In comparison, 27 out of 50 healthy controls (54%) responded. In HSCT recipients treated for BKV-related cystitis, absolute CD4+ T-cell numbers and renal function correlated with BKV-specific cellular responses (p = 0.03 and 0.01, respectively). In one patient, BKV-specific cellular immunity could already be detected at baseline, on day 35 after HSCT and prior to VSTs, and remained increased until day 226 after VSTs (78 vs. 7 spots increment). In conclusion, the ELISpot appears to be suitable to sensitively monitor BKV-specific cellular immunity in HSCT recipients, even early after transplantation or in the long term after VSTs.

Dynamic International Prognostic Scoring System scores, pre-transplant therapy and chronic graft-versus-host disease determine outcome after allogeneic hematopoietic stem cell transplantation for myelofibrosis.

BACKGROUND: Myelofibrosis is a myeloproliferative stem cell disorder curable exclusively by allogeneic hematopoietic stem cell transplantation and is associated with substantial mortality and morbidity. The aim of this study was to assess disease-specific and transplant-related risk factors that influence post-transplant outcome in patients with myelofibrosis. DESIGN AND METHODS: We retrospectively assessed 76 consecutive patients with primary (n=47) or secondary (n=29) myelofibrosis who underwent bone marrow (n=6) or peripheral blood stem cell (n=70) transplantation from sibling (n=30) or unrelated (n=46) donors between January 1994 and December 2010. The median follow-up of surviving patients was 55 ± 7.5 months. RESULTS: Primary graft failure occurred in 5% and the non-relapse mortality rate at 1 year was 28%. The relapse-free survival rate was 50% with a relapse rate of 19% at 5 years. The use of pharmacological pre-treatment and the post-transplant occurrence of chronic graft-versus-host disease were significant independent unfavourable risk factors for post-transplant survival in multivariate analysis. Using the Dynamic International Prognostic Scoring System for risk stratification, low-risk patients had significantly better overall survival (P=0.014, hazard ratio 1.4) and relapse-free survival (P=0.02, hazard ratio 1.3) compared to the other risk groups of patients. The additional inclusion of thrombocytopenia, abnormal karyotype and transfusion need (Dynamic International Prognostic Scoring System Plus) resulted in a predicted 5-year overall survival of 100%, 51%, 54% and 30% for low, intermediate-1, intermediate-2 and high-risk groups, respectively. The relapse incidence was significantly higher in the absence of chronic graft-versus-host disease (P=0.006), and pharmacological pre-treatment (n=43) was associated with reduced relapse-free survival (P=0.001). CONCLUSIONS: The data corroborate a strong correlation between alloreactivity and long-term post-transplant disease control and confirm an inverse relationship between disease stage, pharmacotherapy and outcome after allogeneic hematopoietic stem cell transplantation for myelofibrosis. The Dynamic International Prognostic Scoring System was demonstrated to be useful for risk stratification of patients with myelofibrosis who are to undergo hematopoietic stem cell transplantation.

Use of the activating gene mutation of the tyrosine kinase (VAL617Phe) JAK2 as a minimal residual disease marker in patients with myelofibrosis and myeloid metaplasia after allogeneic stem cell transplantation.

Here we report on the use of a new real-time polymerase chain reaction (PCR) method to detect and quantify the activating gene mutation of the tyrosine kinase JAK2. We evaluated patients with myelofibrosis with myeloid metaplasia (MMM; n=25) for the gene mutation prior to allogeneic stem cell transplantation and monitored them in the long-term follow up of 125 months (median 15, range 4-125) after transplant. The results obtained were correlated to the chimerism status of these patients. The JAK2 gene mutation was detected in 15 of 25 analyzed patients prior to transplant. Three patients who were again positive for JAK2 after transplant also had mixed chimerism status. These three patients relapsed from MMM shortly after JAK2 gene mutation was detected for the first time after transplant. Our presented data shows the feasibility of the detection of JAK2 gene mutation by real-time PCR as a minimal residual disease marker after transplant.

Mutations in innate immune system NOD2/CARD 15 and TLR-4 (Thr399Ile) genes influence the risk for severe acute graft-versus-host disease in patients who underwent an allogeneic transplantation.

BACKGROUND: NOD2 and TLR-4 genes belong to the innate immune system that detects invading pathogens through several pattern-recognition receptors. Here we analyzed 403 patients for NOD2 gene mutations and 307 patients for TLR-4 gene mutations (Thr399Ile) with their respective donors and correlated the results with the incidence of acute graft-versus-host disease (aGVHD), severe acute GVHD (saGVHD), the risk for transplant-related mortality (TRM), overall survival (OS) and incidence of infectious complications. METHODS: We performed a retrospective single-center study. Genotyping of TLR-4 and NOD2 were evaluated by real-time polymerase chain reaction. RESULTS: Surprisingly, we found a significant reduced incidence of aGVHD, saGVHD, and intestinal GVHD for patients with NOD2 gene mutations on the donor side with 50%, 0% and 2% compared to patients with the wild-type NOD2 gene with 65%, 17%, and 26%, respectively (P<0.02). However, the incidence of saGVHD increased in patients with NOD2 mutations on the patient and donor (P/D) side with 44% versus 17% compared to patients with the wild-type gene (P<0.03). TLR-4 gene mutations at P/D side had an increased risk for saGVHD with 42% versus 15% of patients with wild-type gene (P<0.04). OS, TRM, and incidence of infectious complications were not influenced by the mutated genes. Multivariate analysis confirmed that NOD2 gene mutations on the donor side had a reduced risk for saGVHD (P<0.001), whereas mutations of the NOD2 gene on P/D side had an increased risk for saGVHD (P<0.01) in our analysis. CONCLUSIONS: These results suggest that NOD2 mutations have influence on the occurrence of acute GVHD after transplantation.

Reduced risk for molecular disease in patients with chronic myeloid leukemia after transplantation from a KIR-mismatched donor.

BACKGROUND: To examine how killer-cell immunoglobulin-like receptor (KIR) ligand incompatibilities effect molecular relapse (MR), we compared the occurrence of bcr-abl-positive reverse-transcriptase polymerase chain reaction (RT-PCR) results in 236 CML patients (pts) after human leukocyte antigen (HLA)-identical (n=158) (group 1), HLA class I antigen mismatched and KIR-ligand compatible (n=49) (group 2), and HLA class I antigen mismatched and KIR-ligand incompatible (n=29) (group 3) hematopoietic stem-cell transplantation. METHODS: We performed a retrospective single-center study. MR was evaluated using the real-time RT-PCR method for the detection of bcr-abl transcripts. RESULTS: In the first group, 133 of 158 (84%) pts were in the first chronic phase of CML, and the corresponding figures were 33 of 49 (67%) pts in group 2 and 19 of 29 (64%) in group 3 (P<0.05). MR occurred in 1 of 29 (3%) pts in group 3 compared with 62 of 158 (39%) pts in group 1 and in 11 of 49 (22%) pts in group 2 (P<0.001). A hematologic relapse developed in 20 of 158 (13%) pts in group 1, 2 of 49 (4%) pts in group 2, and in 0 of 29 (0%) pts in group 3 (P<0.05). Multivariate analysis confirmed that KIR mismatches are a strong independent predictor for the occurrence of MR after transplantation (P<0.02). The 5-year overall survival rate did not vary greatly between the three groups (67% in group 1, 52% in group 2, and 66% in group 3). CONCLUSIONS: These results suggest that KIR-ligand incompatibility is an important prognostic factor in the occurrence of MR after transplantation for CML.

Maternal molecular features and gene profiling of monocytes during first trimester pregnancy.

We examined the molecular characteristics of monocytes of pregnant and non-pregnant women to investigate the molecular effects that are associated with immunoregulation at the maternal-fetal interface. We analyzed molecular features and target genes in monocytes of pregnant women using flow cytometry, real-time PCR and oligonucleotide microarray technology. CD14(high) monocytes and several immune gene members including CD200, CD200R, IDO, IFI27, IL-10 and G0S2 were found to be differentially expressed in monocytes throughout pregnancy. In addition, transcripts within components of the signaling cascade of immune cells (HLA-DRB4, HBEGF, IL-8, CD3D, CCL5), and of several transcription factors (SOCS1, CXCL10, ID1, ID2) were altered in the monocytes of pregnant women. Further studies will be needed to elucidate the biological significance of our observation.

Modulating impact of human chorionic gonadotropin hormone on the maturation and function of hematopoietic cells.

hCG hormone is a naturally occurring, immune-modulating agent, which is highly expressed during pregnancy and causes improvements of some autoimmune diseases such as multiple sclerosis and Crohn's disease. Little is known about its immune-modulating effects. This study in MNCs of women who received hCG as preconditioning prior to IVF demonstrates that hCG increases anti-inflammatory IL-27 expression and reduces inflammatory IL-17 expression. In addition, we found increased IL-10 levels and elevated numbers of Tregs in peripheral blood of women after hCG application. Rejection of allogeneic skin grafts was delayed in female mice receiving hCG. We conclude that hCG may be useful for the induction of immune tolerance in solid organ transplantation.

Growth factor-independent 1B gene (GFI1B) is overexpressed in erythropoietic and megakaryocytic malignancies and increases their proliferation rate.

Growth factor-independent 1B (GFI1B) is a transcription factor essential for the development and differentiation of erythroid and megakaryocytic lineages. We evaluated the GFI1B expression in erythroleukaemia and megakaryocytic leukaemia, as well as in patients with other subtypes of acute myeloid leukaemia (AML), acute lymphoblastic leukaemia (ALL), chronic myeloid leukaemia (CML), myelodysplastic syndrome (MDS), severe aplastic anaemia (SAA), myelofibrosis with myeloid metaplasia (MMM) and in healthy volunteers. GFI1B expression was increased at least threefold in patients with erythroleukaemia (P < 0.01 compared with controls) and megakaryocytic leukaemia (P < 0.05) as well as in their corresponding leukaemic cell lines HEL, K562, CMK and M-07e. Patients with undifferentiated or monocytic AML, ALL, MMM, MDS and CML had no significantly altered GFI1B expression, whereas GFI1B expression was decreased 10-fold in patients with SAA (P < 0.0001 compared with controls). Silencing GFI1B by transfection with small interfering RNA (siRNA) markedly reduced the proliferation rate in the leukaemic cell lines HEL, K562 and NB4 (P < 0.01). Concomitantly, we observed a two- to threefold increase in the apoptosis rate in these cells after transfection with siRNA towards GFI1B. Our data indicate that GFI1B plays a major role in AML-M6 and AML-M7 and qualifies as a target for anti-leukaemic strategies in these malignancies.

Quantitative analysis of chimerism after allogeneic stem cell transplantation by real-time polymerase chain reaction with single nucleotide polymorphisms, standard tandem repeats, and Y-chromosome-specific sequences.

We compared the results of chimerism analyses with real-time SNP-PCR to those obtained by the classical STR-PCR method in 135 hematopoietic stem cell transplantation recipients. Using 10 different SNP gene loci, the SNP-PCR method was able to discriminate patient from donor cells in 125 of 135 cases (93%), whereas the use of 11 different STR gene loci with the STR-PCR analysis using agarose or polyacrylamide gel resolution resulted in accurate donor-host discrimination in all patients. Of the 470 analyzed samples we found in 74% concordant results for both chimerism methods. In all 26% discordant cases the SNP-chimerism method showed mixed chimerism (MC), whereas the STR-method found complete chimerism (CC). As a consequence, the SNP-PCR chimerism analysis method detected a MC prior to the occurrence of relapse significantly earlier than the STR-PCR chimerism method (120 vs. 30 days, P < 0.007). The probability of relapses was significantly higher in patients with increasing MC (70%) compared to 30% in patients with CC (P < 0.00001) associated with a significantly shorter overall survival in patients with increasing MC. The multivariate Cox model showed that chimerism analsis with SNP-PCR was the only significant risk factor predicting relapse (RR 6.08, P < 0.0001).Furthermore, we analyzed the chimerism status in male recipients with a female donor in 580 samples of 134 patients using quantitative real-time PCR of Y-chromosome-specific sequences and compared the results with interphase XY-fluorescent in situ hybridization (FISH). MC without signs of relapse was detected in 35% of samples using quantitative real-time PCR of Y-chromosome-specific sequences. The detected Y-DNA amounts were low compared to the amounts detected in 104 samples of 42 patients with leukemic relapse at the time of analysis (P < 0.0001). Quantitative real-time PCR of Y-chromosome-specific sequences detected therefore an increasing MC with high residual host DNA amounts approximately 143 days (mean) prior to the occurrence of relapse. By comparing the results of Y-chromosome PCR with the XY-FISH analysis we found concordant results in 73% in patients with myeloablative regimens. The XY-FISH could detect 12 relapses, whereas the Y-chromosome PCR detect 36 relapses by MC (P < 0.005). Residual host cells gradually decreased during the posttransplant period from a mean of 5.4 ng (first months) to 0.5 ng (above 5 years) without evidence of relapses. The probability of relapses was significantly higher in patients with increasing MC (100%) compared to 8% in patients with CC (P < 0.00001) associated with a significantly shorter overall survival in patients with increasing MC. The multivariate Cox model showed that chimerism analysis of Y-chromosome-specific sequences is an important risk factor for relapse (RR 17.0, P < 0.0001). We conclude that the use of real-time SNP or Y-PCR may be superior to the STR-PCR or interphase XY-FISH methods in detecting patients who are at high risk for relapse after transplant.