Clonal relapse dynamics in acute myeloid leukemia following allogeneic hematopoietic cell transplantation.

ABSTRACT: Patients with acute myeloid leukemia (AML) who experience relapse following allogeneic hematopoietic cell transplantation (alloHCT) face unfavorable outcomes regardless of the chosen relapse treatment. Early detection of relapse at the molecular level by measurable residual disease (MRD) assessment enables timely intervention, which may prevent hematological recurrence of the disease. It remains unclear whether molecular MRD assessment can detect MRD before impending relapse and, if so, how long in advance. This study elucidates the molecular architecture and kinetics preceding AML relapse by using error-corrected next-generation sequencing (NGS) in 74 patients with AML relapsing after alloHCT, evaluating 140 samples from peripheral blood collected 0.6 to 14 months before relapse. At least 1 MRD marker became detectable in 10%, 38%, and 64% of patients at 6, 3, and 1 month before relapse, respectively. By translating these proportions into monitoring intervals, 38% of relapses would have been detected through MRD monitoring every 3 months, whereas 64% of relapses would have been detected with monthly intervals. The relapse kinetics after alloHCT are influenced by the functional class of mutations and their stability during molecular progression. Notably, mutations in epigenetic modifier genes exhibited a higher prevalence of MRD positivity and greater stability before relapse, whereas mutations in signaling genes demonstrated a shorter lead time to relapse. Both DTA (DNMT3A, TET2, and ASXL1) and non-DTA mutations displayed similar relapse kinetics during the follow-up period after alloHCT. Our study sets a framework for MRD monitoring after alloHCT by NGS, supporting monthly monitoring from peripheral blood using all variants that are known from diagnosis.

The graft-versus-leukemia effect of prophylactic donor lymphocyte infusions after allogeneic stem cell transplantation is equally effective in relapse prevention but safer compared to spontaneous graft-versus-host disease.

Therapeutic donor lymphocyte infusions (tDLI) are used to reinforce the graft-versus-leukemia (GvL) effect in relapse after allogeneic stem cell transplantation (alloSCT). In contrast, the role of prophylactic DLI (proDLI) in preventing leukemia relapse has been less clearly established, although supported by retrospective, case-control, and registry analyses. We report a prospective, monocentric, ten year cohort of patients with high risk acute leukemias (AL) or myelodysplasia (MDS) in whom proDLI were applied beyond day +120 post alloSCT to compensate for lack of GvL.272 consecutive allotransplanted AL or MDS patients in complete remission and off immunosuppression at day +120 were stratified according to the prior appearance of relevant GvHD (acute GvHD °II-IV or extensive chronic GvHD) as a clinical indicator for GvL. Escalating doses of unmodified proDLI were applied to 72/272 patients without prior relevant GvHD. Conversely, 157/272 patients with prior spontaneous GvHD did not receive proDLI, nor did 43/272 patients with contraindications (uncontrolled infections, patient refusal, DLI unavailability).By day 160-landmark analysis (median day of first DLI application), proDLI recipients had significantly higher five-year overall (OS) and disease free survival (DFS) (77% and 67%) than patients with spontaneous GvHD (54% and 53%) or with contraindications (46% and 45%) (p=0.003). Relapse incidence for patients with proDLI (30%) or spontaneous GvHD (29%) was significantly lower than in patients with contraindications (39%; p=0.021). With similar GvHD incidence beyond day +160, non-relapse mortality (NRM) was less with proDLI (5%) than without proDLI (18%; p=0.036).In conclusion, proDLI may be able to compensate for lack of GvL in alloSCT recipients with high risk AL or MDS.

Measurable residual disease monitoring by NGS before allogeneic hematopoietic cell transplantation in AML.

Molecular measurable residual disease (MRD) assessment is not established in approximately 60% of acute myeloid leukemia (AML) patients because of the lack of suitable markers for quantitative real-time polymerase chain reaction. To overcome this limitation, we established an error-corrected next-generation sequencing (NGS) MRD approach that can be applied to any somatic gene mutation. The clinical significance of this approach was evaluated in 116 AML patients undergoing allogeneic hematopoietic cell transplantation (alloHCT) in complete morphologic remission (CR). Targeted resequencing at the time of diagnosis identified a suitable mutation in 93% of the patients, covering 24 different genes. MRD was measured in CR samples from peripheral blood or bone marrow before alloHCT and identified 12 patients with persistence of an ancestral clone (variant allele frequency [VAF] >5%). The remaining 96 patients formed the final cohort of which 45% were MRD+ (median VAF, 0.33%; range, 0.016%-4.91%). In competing risk analysis, cumulative incidence of relapse (CIR) was higher in MRD+ than in MRD- patients (hazard ratio [HR], 5.58; P < .001; 5-year CIR, 66% vs 17%), whereas nonrelapse mortality was not significantly different (HR, 0.60; P = .47). In multivariate analysis, MRD positivity was an independent negative predictor of CIR (HR, 5.68; P < .001), in addition to FLT3-ITD and NPM1 mutation status at the time of diagnosis, and of overall survival (HR, 3.0; P = .004), in addition to conditioning regimen and TP53 and KRAS mutation status. In conclusion, NGS-based MRD is widely applicable to AML patients, is highly predictive of relapse and survival, and may help refine transplantation and posttransplantation management in AML patients.

Characterization of High-Avidity Cytomegalovirus-Specific T Cells with Differential Tetramer Binding Coappearing after Allogeneic Stem Cell Transplantation.

CMV reactivation is a major complication after allogeneic stem cell transplantation (SCT). Immune reconstitution of CMV-specific CTLs (CMV-CTLs) is essential for virus control. During CMV-CTL monitoring using mutated HLA/CMV tetramers selectively detecting high-avidity T cells, we observed coappearance of CMV-CTLs with low (CMV tetlow CTLs) and high tetramer binding (CMV tethigh CTLs) in 53/115 CMV IgG+ patients stem cell transplanted from CMV IgG+ donors. However, the relevance of these coappearing differentially tetramer binding ("dual") CMV-CTLs was unclear. In this study, we investigated the kinetics, properties, and clinical impact of coappearing CMV tetlow and tethigh CTLs after allogeneic SCT. Patients with dual CMV-CTLs had more CMV tethigh than tetlow CTLs. Chimerism analysis of isolated CMV tetlow and tethigh CTLs revealed their exclusive donor origin. CMV tetlow and tethigh CTLs had an identical effector memory CD45RA-CCR7- and CD45RA+CCR7- T cell distribution, equal differentiation, senescence, and exhaustion marker expression and were negative for regulatory CD8+ T cell markers. Isolated CMV tetlow and tethigh CTLs were equally sensitive to CMV peptides in IFN-γ release and cytotoxicity assays. However, CMV tethigh CTLs proliferated more in response to low CMV peptide concentrations than tetlow CTLs. TCR repertoire analysis revealed that CMV tetlow and tethigh CTLs use different TCRs. Finally, dual CMV-CTLs were not associated with CMV antigenemia. In conclusion, these data show for the first time, to our knowledge, that both CMV tetlow and tethigh CTLs are functional effector T cells differing by proliferation, numbers in peripheral blood, and probably by their precursors without increasing the CMV reactivation risk after allogeneic SCT.

Possible Impact of Cytomegalovirus-Specific CD8+ T Cells on Immune Reconstitution and Conversion to Complete Donor Chimerism after Allogeneic Stem Cell Transplantation.

Complete donor chimerism is strongly associated with complete remission after allogeneic stem cell transplantation (allo-SCT) in patients with hematologic malignancies. Donor-derived allo-immune responses eliminate the residual host hematopoiesis and thereby mediate the conversion to complete donor chimerism. Recently, cytomegalovirus (CMV) reactivation was described to enhance overall T cell reconstitution, to increase graft-versus-host disease incidence, and to reduce the leukemia relapse risk. However, the link between CMV and allo-immune responses is still unclear. Here, we studied the relationship between CMV-specific immunity, overall T cell reconstitution, and residual host chimerism in 106 CMV-seropositive patients transplanted after reduced-intensity conditioning including antithymocyte globulin. In accordance with previous reports, the recovery of CMV-specific cytotoxic T cells (CMV-CTLs) was more frequent in CMV-seropositive recipients (R) transplanted from CMV-seropositive than from seronegative donors (D). However, once CMV-CTLs were detectable, the reconstitution of CMV-specific CTLs was comparable in CMV R+/D- and R+/D+ patients. CD3+ and CD8+ T cell reconstitution was significantly faster in patients with CMV-CTLs than in patients without CMV-CTLs both in the CMV R+/D- and R+/D+ setting. Moreover, CMV-CTL numbers correlated with CD3+ and CD8+ T cell numbers in both settings. Finally, presence of CMV-CTLs was associated with low host chimerism levels 3 months after allo-SCT. In conclusion, our data provide a first indication that CMV-CTLs in CMV-seropositive patients might trigger the reconstitution of T cells and allo-immune responses reflected by the conversion to complete donor chimerism.

IL-7 receptor expression identifies suicide gene-modified allospecific CD8+ T cells capable of self-renewal and differentiation into antileukemia effectors.

In allogeneic hematopoietic cell transplantation (HSCT), donor T lymphocytes mediate the graft-versus-leukemia (GVL) effect, but induce graft-versus-host disease (GVHD). Suicide gene therapy-that is, the genetic induction of a conditional suicide phenotype into donor T cells-allows dissociating the GVL effect from GVHD. Genetic modification with retroviral vectors after CD3 activation reduces T-cell alloreactivity. We recently found that alloreactivity is maintained when CD28 costimulation, IL-7, and IL-15 are added. Herein, we used the minor histocompatibility (mH) antigens HA-1 and H-Y as model alloantigens to directly explore the antileukemia efficacy of human T cells modified with the prototypic suicide gene herpes simplex virus thymidine kinase (tk) after activation with different stimuli. Only in the case of CD28 costimulation, IL-7, and IL-15, the repertoire of tk(+) T cells contained HA-1- and H-Y-specific CD8(+) cytotoxic T cells (CTL) precursors. Thymidine kinase-positive HA-1- and H-Y-specific CTLs were capable of self-renewal and differentiation into potent antileukemia effectors in vitro, and in vivo in a humanized mouse model. Self-renewal and differentiation coincided with IL-7 receptor expression. These results pave the way to the clinical investigation of T cells modified with a suicide gene after CD28 costimulation, IL-7, and IL-15 for a safe and effective GVL effect.

Peptide length extension skews the minor HA-1 antigen presentation toward activated dendritic cells but reduces its presentation efficiency.

Minor histocompatibility Ags (mHags) are important targets of the graft-versus-leukemia effect after HLA-matched allogeneic stem cell transplantation. mHags are HLA-restricted polymorphic peptides expressed on normal and leukemia cells. Vaccination with hematopoiesis-restricted mHag peptides, such as HA-1, may boost the graft-versus-leukemia effect. However, some animal studies indicate that peptides exactly reflecting immunogenic T cell epitopes (short peptides [SPs]) induce tolerance that is potentially due to systemic Ag spreading. Peptide length extension (long peptides [LPs]) may optimize immune responses by restricting and prolonging Ag presentation on dendritic cells (DCs). In this study, we compared the in vitro characteristics and T cell-stimulatory capacities of a human 30-mer HA-1 LP with the 9-mer HA-1 SP. DCs presented the HA-1 LP and SP and expanded HA-1-specific cytotoxic T cell lines. As hypothesized, HA-1 LP presentation, but not SP presentation, was largely restricted to activated DCs and was nearly absent on other hematopoietic cells. However, DCs presented the HA-1 LP 2-3 log levels less efficiently than the SP. Finally, the decay of HA-1 LP and SP presentation on DCs was comparable. We conclude that HA-1 LP and SP differ in their in vitro characteristics and that only comparative clinical studies after allogeneic stem cell transplantation may reveal the optimal HA-1 vaccine.

Navigating preemptive and therapeutic donor lymphocyte infusions in advanced myeloid malignancies by high-sensitivity chimerism analysis.

Preemptive and therapeutic donor lymphocyte infusions (preDLI and tDLI) are widely used in relapsing and relapsed hematopoietic malignancies after allogeneic stem cell transplantation (alloSCT) to enhance the graft-versus-malignancy effect. However, in advanced myeloid malignancies, long-term survival after preDLI and tDLI remains low, reflecting our inability to master the double-edged sword of alloreactivity, balancing anti-neoplastic activity versus graft-versus-host disease (GvHD). We previously evaluated a quantitative PCR-based high-sensitivity chimerism (hs-chimerism) based on insertion/deletion polymorphisms instead of short tandem repeats, where increasing host chimerism in peripheral blood predicts relapse more than a month before clinical diagnosis, and declining host chimerism signals anti-host alloreactivity. Here we report 32 consecutive patients with advanced myeloid malignancies receiving preDLI or tDLI "navigated" by hs-chimerism ("navigated DLI"). We compared them to a historical cohort of 110 consecutive preDLI or tDLI recipients, prior to implementation of hs-chimerism at our institution ("controls"). Both groups were comparable regarding age, gender, conditioning, donor type, and time to DLI. With longer median follow-up of the navigated DLI group (8.5 versus 5 months), their landmark overall (64%) and disease-free survival (62%) at 2 years from first DLI compared favorably with controls (23% and 21%, respectively). Improved survival of navigated DLI was due to both reduced relapse incidence (38% versus 60%) and non-relapse mortality (17% versus 44%) at 2 years. Early relapse prediction by hs-chimerism allowed a preemptive approach in 28% of navigated DLI versus 7% in controls. Our results confirm hs-chimerism as a highly valuable tool for monitoring and steering immune interventions after alloSCT.

Hypomethylating drugs convert HA-1-negative solid tumors into targets for stem cell-based immunotherapy.

Clinical responses of solid tumors after allogeneic human leukocyte antigen-matched stem cell transplantation (SCT) often coincide with severe graft-versus-host disease (GVHD). Targeting minor histocompatibility antigens (mHags) with hematopoiesis- and cancer-restricted expression, for example, HA-1, may allow boosting the antitumor effect of allogeneic SCT without risking severe GVHD. The mHag HA-1 is aberrantly expressed in cancers of most entities. However, an estimated 30% to 40% of solid tumors do not express HA-1 (ie, are HA-1(neg)) and cannot be targeted by HA-1-specific immunotherapy. Here, we investigated the transcriptional regulation of HA-1 gene expression in cancer. We found that DNA hypermethylation in the HA-1 promoter region is closely associated with the absence of HA-1 gene expression in solid tumor cell lines. Moreover, we detected HA-1 promoter hypermethylation in primary cancers. The hypomethylating agent 5-aza-2'-deoxycytidine induced HA-1 expression only in HA-1(neg) tumor cells and sensitized them for recognition by HA-1-specific cytotoxic T lymphocytes. Contrarily, the histone deacetylation inhibitor trichostatin A induced HA-1 expression both in some HA-1(neg) tumor cell lines and in normal nonhematopoietic cells. Our data suggest that promoter hypermethylation contributes to the HA-1 gene regulation in tumors. Hypomethylating drugs might extend the safe applicability of HA-1 as an immunotherapeutic target on solid tumors after allogeneic SCT.

Severe allo-immune antibody-associated peripheral and central nervous system diseases after allogeneic hematopoietic stem cell transplantation.

Allogeneic hematopoietic stem cell transplantation (alloHSCT) is a curative treatment for hematologic malignancies. Acute and chronic graft-versus-host disease (GvHD) are the major immune-mediated complications after alloHSCT. However, there is controversy whether neurologic complications after alloHSCT might represent manifestations of GvHD. We report three patients who acquired distinct, severe immune-mediated peripheral or central nervous system diseases after alloHSCT without other, concomitant GvHD manifestations. One patient had been diagnosed with B-cell chronic lymphocytic leukemia and two patients with high risk myelodysplastic syndrome. Patient #1 presented as LGI1- and GAD-IgG positive immune-mediated encephalitis, patient #2 was diagnosed with MOG-IgG positive encephalomyelitis, and patient #3 had chronic inflammatory polyneuropathy associated with SSA(Ro)-IgG positive Sjögren's syndrome. 100% donor chimerism was detectable in the peripheral blood in all three. The specific antibodies were undetectable in donors' and patients' blood before alloHSCT suggesting that the antibodies had arisen from the transplanted donor immune system. Early intensive immunotherapy led to improvement of clinical symptoms and stability of the neurological disease, however, at the cost of losing the graft-versus-malignancy effect in one patient. In conclusion, we provide evidence of isolated, severe allo-immune diseases of the peripheral and central nervous system as complications of alloHSCT ("neuro-GvHD"). Interdisciplinary surveillance and thorough diagnostic work-up are needed for early diagnosis and treatment of neuro-immunologic complications after alloHSCT to improve the otherwise poor outcome.

Clonal expansion of CD8+ T cells reflects graft-versus-leukemia activity and precedes durable remission following DLI.

Donor lymphocyte infusion (DLI) is a standard of care for relapse of acute myeloid leukemia after allogeneic hematopoietic stem cell transplantation. Currently it is poorly understood how and when CD8+ αß T cells exert graft-versus-leukemia (GVL) activity after DLI. Also, there is no reliable biomarker to monitor GVL activity of the infused CD8+ T cells. Therefore, we analyzed the dynamics of CD8+ αß T-cell clones in patients with DLI. In this prospective clinical study of 29 patients, we performed deep T-cell receptor ß (TRB ) sequencing of sorted CD8+ αß T cells to track patients' repertoire changes in response to DLI. Upon first occurrence of GVL, longitudinal analyses revealed a preferential expansion of distinct CD8+TRB clones (n = 14). This did not occur in samples of patients without signs of GVL (n = 11). Importantly, early repertoire changes 15 days after DLI predicted durable remission for the 36-month study follow-up. Furthermore, absence of clonal outgrowth of the CD8+TRB repertoire after DLI was an early biomarker that predicted relapse at a median time of 11.2 months ahead of actual diagnosis. Additionally, unbiased sample analysis regardless of the clinical outcome revealed that patients with decreasing CD8+TRB diversity at day 15 after DLI (n = 13) had a lower relapse incidence (P = .0040) compared with patients without clonal expansion (n = 6). In conclusion, CD8+TRB analysis may provide a reliable tool for predicting the efficacy of DLI and holds the potential to identify patients at risk for progression and relapse after DLI.

Posttransplantation MRD monitoring in patients with AML by next-generation sequencing using DTA and non-DTA mutations.

Next-generation sequencing (NGS)-based measurable residual disease (MRD) monitoring in patients with acute myeloid leukemia (AML) is widely applicable and prognostic prior to allogeneic hematopoietic cell transplantation (alloHCT). We evaluated the prognostic role of clonal hematopoiesis-associated DNMT3A, TET2, and ASXL1 (DTA) and non-DTA mutations for MRD monitoring post-alloHCT to refine MRD marker selection. Of 154 patients with AML, 138 (90%) had at least one mutation at diagnosis, which were retrospectively monitored by amplicon-based error-corrected NGS on day 90 and/or day 180 post-alloHCT. MRD was detected in 34 patients on day 90 and/or day 180 (25%). The rate of MRD positivity was similar when DTA and non-DTA mutations were considered separately (17.6% vs 19.8%). DTA mutations had no prognostic impact on cumulative incidence of relapse, relapse-free survival, or overall survival in our study and were removed from further analysis. In the remaining 131 patients with at least 1 non-DTA mutation, clinical and transplantation-associated characteristics were similarly distributed between MRD-positive and MRD-negative patients. In multivariate analysis, MRD positivity was an independent adverse predictor of cumulative incidence of relapse, relapse-free survival, and overall survival but not of nonrelapse mortality. The prognostic effect was independent of different cutoffs (above limit of detection, 0.1% and 1% variant allele frequency). MRD log-reduction between diagnosis and post-alloHCT assessment had no prognostic value. MRD status post-alloHCT had the strongest impact in patients who were MRD positive prior to alloHCT. In conclusion, non-DTA mutations are prognostic NGS-MRD markers post-alloHCT, whereas the prognostic role of DTA mutations in the posttransplant setting remains open.

A multicenter prospective, randomized, placebo-controlled phase II/III trial for preemptive acute graft-versus-host disease therapy.

Acute graft-versus-host disease (aGvHD) contributes to about 50% of transplant-related mortality (non-relapse mortality) after allogeneic hematopoietic stem cell transplantation (HSCT). Here the predictive value of a urinary proteomic profile (aGvHD_MS17) was tested together with preemptive prednisolone therapy. Two-hundred and fifty-nine of 267 patients were eligible for analysis. Ninety-two patients were randomized upon aGvHD_MS17 classification factor above 0.1 to receive either prednisolone (2-2.5 mg/kg, N = 44) or placebo (N = 47; N = 1 randomization failure) for 5 days followed by tapering. The remaining 167 patients formed the observation group. The primary endpoint of the randomized trial was incidence of aGvHD grade II between randomization and day +100 post HSCT. Analysis of the short-term preemptive prednisolone therapy in the randomized patients showed no significant difference in incidence or severity of acute GvHD (HR: 1.69, 95% CI: 0.66-4.32, P = 0.27). Prednisolone as preemptive treatment did not lead to an increase in relapse (20.2% in the placebo and 14.0% in the prednisolone group (P = 0.46)). The frequency of adverse events was slightly higher in the placebo group (64.4% versus 50%, respectively). Taken together, the results of the Pre-GvHD trial demonstrated the feasibility and safety of preemptive prednisolone treatment in the randomized patients.

Targeting a single mismatched minor histocompatibility antigen with tumor-restricted expression eradicates human solid tumors.

Regressions of metastatic solid tumors after allogeneic human leukocyte antigen (HLA)-matched stem cell transplantation (SCT) are often associated with detrimental graft-versus-host disease (GVHD). The graft-versus-host reaction of the HLA-matched donor is directed mainly against the multiple mismatched minor histocompatibility antigens (mHags) of the patient. mHags are strong HLA-restricted alloantigens with differential tissue distribution. Ubiquitously expressed mHags are the prime in situ targets of GVHD. The mHag HA-1 is hematopoiesis restricted, but displays additionally an aberrant expression on solid tumors. Thus, HA-1 might be an excellent target to boost the anti-solid tumor effect of allogeneic SCT without inducing severe GVHD. Here, we show that cytotoxic T lymphocytes (CTLs) solely targeting the human mHag HA-1 are capable of eradicating 3-dimensional human solid tumors in a highly mHag-specific manner in vitro, accompanied by interferon-gamma release. In vivo, HA-1-specific CTLs distribute systemically and prevent human breast cancer metastases in immunodeficient mice. Moreover, HA-1-specific CTLs infiltrate and inhibit the progression of fully established metastases. Our study provides the first proof for the efficacy of a clinically applicable concept to exploit single mismatched mHags with hematopoiesis- and solid tumor-restricted expression for boosting the anti-solid tumor effect of allogeneic SCT.

Cytomegalovirus-specific CD8+ T-cells are associated with a reduced incidence of early relapse after allogeneic stem cell transplantation.

Leukemia relapse is the main cause for mortality after allogeneic stem cell transplantation (allo-SCT). Donor-derived allo-immune responses eliminate the residual host hematopoiesis and protect against relapse. Cytomegalovirus (CMV) reactivation (CMV-R) after allo-SCT may trigger anti-leukemic effects. The impact of CMV-specific CD8+ T-cells (CMV-CTLs) on the outcome after allo-SCT is currently unknown. Here, we studied the relationship between CMV-CTLs, overall T-cell reconstitution and relapse incidence in 103 patients with acute leukemia (n = 91) or myelodysplastic syndrome (n = 12) following CMV-seropositive recipient/donor (R+/D+) allo-SCT. Patients were subdivided based on the presence or absence of CMV-CTLs at 3 months after allo-SCT. Presence of CMV-CTLs was associated with preceding CMV-R and a fast T-cell reconstitution. Univariate analysis showed a significantly lower 1-, 2- and 5-year cumulative incidence of relapse (CIR) in patients with CMV-CTLs compared to those without CMV-CTLs. Multivariable regression analysis of the outcome performed with other relevant parameters chosen from univariate analysis revealed that presence of CMV-CTLs and chronic graft-versus-host disease (cGvHD) were the only independent factors associated with a low CIR. Onset of relapse was significantly later in patients with CMV-CTLs (median 489 days) than in in those without (median 152 days, p = 0.041) during a five-year follow-up. Presence of CMV-CTLs was associated with a lower incidence of early relapses (1 and 2-years), while cGvHD lead to a lower incidence of late relapses (2 to 5-years). In conclusion, our data show that CMV-CTLs indicate a functional immune-reconstitution protective against early relapse.

Immunotherapy of cancer through targeting of minor histocompatibility antigens.

Minor histocompatibility antigens are allogeneic targets of T-cell mediated graft-versus-tumour effects following allogeneic stem cell transplantation. Recent research has identified several minor histocompatibility antigens as tumour proteins and has also disclosed their unique properties in both the induction and the effector phase of graft-versus-tumour effects. Targeting tumour-specific minor histocompatibility antigens by adoptive immunotherapy will battle against tumour tolerance and evoke allo-immune responses, thereby enhancing graft-versus-tumour effects against leukaemia and solid tumours. Recently acquired knowledge of the role of donor immunisation status, new techniques in the generation of minor histocompatibility antigen-specific cytotoxic T lymphocytes in vitro, and innovative principles in vaccination will help to design clinical trials that exploit minor histocompatibility antigens in the immunotherapy of cancer.

Diagnostic value of highly-sensitive chimerism analysis after allogeneic stem cell transplantation.

Conventional analysis of host chimerism (HC) frequently fails to detect relapse before its clinical manifestation in patients with hematological malignancies after allogeneic stem cell transplantation (allo-SCT). Quantitative PCR (qPCR)-based highly-sensitive chimerism analysis extends the detection limit of conventional (short tandem repeats-based) chimerism analysis from 1 to 0.01% host cells in whole blood. To date, the diagnostic value of highly-sensitive chimerism analysis is hardly defined. Here, we applied qPCR-based chimerism analysis to 901 blood samples of 71 out-patients with hematological malignancies after allo-SCT. Receiver operating characteristics (ROC) curves were calculated for absolute HC values and for the increments of HC before relapse. Using the best cut-offs, relapse was detected with sensitivities of 74 or 85% and specificities of 69 or 75%, respectively. Positive predictive values (PPVs) were only 12 or 18%, but the respective negative predictive values were 98 or 99%. Relapse was detected median 38 or 45 days prior to clinical diagnosis, respectively. Considering also durations of steadily increasing HC of more than 28 days improved PPVs to more than 28 or 59%, respectively. Overall, highly-sensitive chimerism analysis excludes relapses with high certainty and predicts relapses with high sensitivity and specificity more than a month prior to clinical diagnosis.

Risk assessment in haematopoietic stem cell transplantation: minor histocompatibility antigens.

Minor histocompatibility (H) antigens are key molecules in graft-versus-host disease (GvHD) and the graft-versus-tumour effect after allogeneic stem-cell transplantation (SCT). Today, molecular typing methods allow an easy assessment of differences in minor H antigens between patient and donors, so that the GvHD risk in individual patients can be estimated. However, the large number of minor H antigens prevents matching for them to avoid GvHD. Interestingly, mismatching for minor H antigens might improve the outcome of allogeneic SCT. Some minor H antigens are expressed mainly by malignant cells and can therefore serve as excellent targets for cancer immunotherapy. Thus, mismatching for tumour-expressed minor H antigens allows boosting of the curative effect of allogeneic SCT. Current research is elucidating the impact of e.g. donor immunization, immunodominance, or functional expression of minor H antigens on the extent of the GvH response.

Human CD8+ CD57- TEMRA cells: Too young to be called "old".

End-stage differentiation of antigen-specific T-cells may precede loss of immune responses against e.g. viral infections after allogeneic stem cell transplantation (SCT). Antigen-specific CD8+ T-cells detected by HLA/peptide multimers largely comprise CD45RA-/CCR7- effector memory (TEM) and CD45RA+/CCR7- TEMRA subsets. A majority of terminally differentiated T-cells is considered to be part of the heterogeneous TEMRA subset. The senescence marker CD57 has been functionally described in memory T-cells mainly composed of central memory (TCM) and TEM cells. However, its role specifically in TEMRA cells remained undefined. Here, we investigated the relevance of CD57 to separate human CD8+ TEMRA cells into functionally distinct subsets. CD57- CD8+ TEMRA cells isolated from healthy donors had considerably longer telomeres and showed significantly more BrdU uptake and IFN-γ release upon stimulation compared to the CD57+ counterpart. Cytomegalovirus (CMV) specific T-cells isolated from patients after allogeneic SCT were purified into CD57+ and CD57- TEMRA subsets. CMV specific CD57- TEMRA cells had longer telomeres and a considerably higher CMV peptide sensitivity in BrdU uptake and IFN-γ release assays compared to CD57+ TEMRA cells. In contrast, CD57+ and CD57- TEMRA cells showed comparable peptide specific cytotoxicity. Finally, CD57- CD8+ TEMRA cells partially changed phenotypically into TEM cells and gained CD57 expression, while CD57+ CD8+ TEMRA cells hardly changed phenotypically and showed considerable cell death after in vitro stimulation. To the best of our knowledge, these data show for the first time that CD57 separates CD8+ TEMRA cells into a terminally differentiated CD57+ population and a so far functionally undescribed "young" CD57- TEMRA subset with high proliferative capacity and differentiation plasticity.