Matthias Wölfl

An early-biomarker algorithm predicts lethal graft-versus-host disease and survival

BACKGROUND. No laboratory test can predict the risk of nonrelapse mortality (NRM) or severe graft-versus-host disease (GVHD) after hematopoietic cellular transplantation (HCT) prior to the onset of GVHD symptoms. METHODS. Patient blood samples on day 7 after HCT were obtained from a multicenter set of 1,287 patients, and 620 samples were assigned to a training set. We measured the concentrations of 4 GVHD biomarkers (ST2, REG3α, TNFR1, and IL-2Rα) and used them to model 6-month NRM using rigorous cross-validation strategies to identify the best algorithm that defined 2 distinct risk groups. We then applied the final algorithm in an independent test set (n = 309) and validation set (n = 358). RESULTS. A 2-biomarker model using ST2 and REG3α concentrations identified patients with a cumulative incidence of 6-month NRM of 28% in the high-risk group and 7% in the low-risk group (P < 0.001). The algorithm performed equally well in the test set (33% vs. 7%, P < 0.001) and the multicenter validation set (26% vs. 10%, P < 0.001). Sixteen percent, 17%, and 20% of patients were at high risk in the training, test, and validation sets, respectively. GVHD-related mortality was greater in high-risk patients (18% vs. 4%, P < 0.001), as was severe gastrointestinal GVHD (17% vs. 8%, P < 0.001). The same algorithm can be successfully adapted to define 3 distinct risk groups at GVHD onset. CONCLUSION. A biomarker algorithm based on a blood sample taken 7 days after HCT can consistently identify a group of patients at high risk for lethal GVHD and NRM. FUNDING. The National Cancer Institute, American Cancer Society, and the Doris Duke Charitable Foundation.

Rituximab Treatment before Reduced-Intensity Conditioning Transplantation Associates with a Decreased Incidence of Extensive Chronic GVHD

Chronic graft-versus-host-disease (cGVHD) is the major cause of late morbidity and mortality after allogeneic stem cell transplantation. B cells have been reported to be involved in mediating cGVHD. To assess whether preemptive host B cell depletion prevents extensive cGVHD after allogeneic reduced-intensity conditioning transplantation (RICT), 173 patients treated with RICT for various hematologic diseases, who had or had not received Rituximab (Rtx) within 6 month prior to RICT, were analyzed retrospectively. Rtx treatment within 6 months prior to RICT reduced extensive cGVHD significantly from 45.8% to 20.1%. We hypothesize that most likely host B cells initiate cGVHD, and thus, host B cell depletion prior to RICT by Rtx might be a valuable strategy to reduce extensive cGVHD after RICT.

Antigen-specific activation and cytokine-facilitated expansion of naive, human CD8+ T cells.

Antigen-specific priming of human, naive T cells has been difficult to assess. Owing to the low initial frequency in the naive cell pool of specific T cell precursors, such an analysis has been obscured by the requirements for repeated stimulations and prolonged culture time. In this protocol, we describe how to evaluate antigen-specific priming of CD8+ cells 10 d after a single specific stimulation. The assay provides reference conditions, which result in the expansion of a substantial population of antigen-specific T cells from the naive repertoire. Various conditions and modifications during the priming process (e.g., testing new cytokines, co-stimulators and so on) can now be directly compared with the reference conditions. Factors relevant to achieving effective priming include the dendritic cell preparation, the T cell preparation, the cell ratio at the time of priming, the serum source used for the experiment and the timing of addition and concentration of the cytokines used for expansion. This protocol is relevant for human immunology, vaccine biology and drug development.

International, Multicenter Standardization of Acute Graft-versus-Host Disease Clinical Data Collection: A Report from the Mount Sinai Acute GVHD International Consortium.

Acute graft-versus-host disease (GVHD) remains a leading cause of morbidity and nonrelapse mortality after allogeneic hematopoietic cell transplantation. The clinical staging of GVHD varies greatly between transplant centers and is frequently not agreed on by independent reviewers. The lack of standardized approaches to handle common sources of discrepancy in GVHD grading likely contributes to why promising GVHD treatments reported from single centers have failed to show benefit in randomized multicenter clinical trials. We developed guidelines through international expert consensus opinion to standardize the diagnosis and clinical staging of GVHD for use in a large international GVHD research consortium. During the first year of use, the guidance followed discussion of complex clinical phenotypes by experienced transplant physicians and data managers. These guidelines increase the uniformity of GVHD symptom capture, which may improve the reproducibility of GVHD clinical trials after further prospective validation.

Development and validation of a fully GMP-compliant production process of autologous, tumor-lysate-pulsed dendritic cells

BACKGROUND AND AIMS One of the major challenges of dendritic cell (DC) vaccination is the establishment of harmonized DC production protocols. Here, we report the transfer and validation of a successfully used open DC manufacturing method into a closed system, good manufacturing practice (GMP)-compatible protocol. METHODS All production steps (lysate generation, monocyte selection, DC culture and cryopreservation) were standardized and validated. RESULTS Tumor lysate was characterized by histology, mechanically homogenized and avitalized. This preparation yielded a median of 58 ± 21 μg protein per milligram of tumor tissue. Avitality was determined by trypan blue staining and confirmed in an adenosine triphosphate release assay. Patient monocytes were isolated by elutriation or CD14 selection, which yielded equivalent results. DCs were subsequently differentiated in Teflon bags for an optimum of 7 days in CellGro medium supplemented with interleukin (IL)-4 and granulocyte macrophage colony stimulating factor and then matured for 48 h in tumor necrosis factor-α and IL-1ß after pulsing with tumor lysate. This protocol resulted in robust and reproducible upregulation of DC maturation markers such as cluster of differentiation (CD)80, CD83, CD86, human leukocyte antigen-DR and DC-SIGN. Functionality of these DCs was shown by directed migration toward C-C motif chemokine ligand 19/21, positive T-cell stimulatory capacity and the ability to prime antigen-specific T cells from naive CD8(+) T cells. Phenotype stability, vitality and functionality of DCs after cryopreservation, thawing and washing showed no significant loss of function. Comparison of clinical data from 146 patients having received vaccinations with plate-adherence versus GMP-grade DCs showed no inferiority of the latter. CONCLUSIONS Our robust, validated and approved protocol for DC manufacturing forms the basis for a harmonized procedure to produce cancer vaccines, which paves the way for larger multi-center clinical trials.

MB3W1 is an orthotopic xenograft model for anaplastic medulloblastoma displaying cancer stem cell- and Group 3-properties.

BackgroundMedulloblastoma is the most common malignant brain tumor in children and can be divided in different molecular subgroups. Patients whose tumor is classified as a Group 3 tumor have a dismal prognosis. However only very few tumor models are available for this subgroup.MethodsWe established a robust orthotopic xenograft model with a cell line derived from the malignant pleural effusions of a child suffering from a Group 3 medulloblastoma.ResultsBesides classical characteristics of this tumor subgroup, the cells display cancer stem cell characteristics including neurosphere formation, multilineage differentiation, CD133/CD15 expression, high ALDH-activity and high tumorigenicity in immunocompromised mice with xenografts exactly recapitulating the original tumor architecture.ConclusionsThis model using unmanipulated, human medulloblastoma cells will enable translational research, specifically focused on Group 3 medulloblastoma.

Impact of Sequence Variation in a Dominant HLA-A*02-Restricted Epitope in Hepatitis C Virus on Priming and Cross-Reactivity of CD8+ T Cells

ABSTRACT CD8+ T cells are an essential component of successful adaptive immune responses against hepatitis C virus (HCV). A major obstacle to vaccine design against HCV is its inherent viral sequence diversity. Here, we test the hypothesis that different sequence variants of an immunodominant CD8+ T cell epitope, all binding with high affinity to HLA class I, target different T cell receptor repertoires and thereby influence the quality of the CD8+ T cell response. The impacts of sequence differences in the HLA-A*02-restricted HCV NS31406–1415 epitope on in vitro priming of naive CD8+ T cells from seronegative donors and cross-reactivity of primed T cells with other epitope variants were characterized. Although the six epitope variants tested were all high-affinity binders to HLA-A*02:01, substantial differences in priming and cross-reactivity of CD8+ T cells were observed. The variant associated with the most reproducible priming and induction of T cells with broad cross-reactivity was a genotype 1b variant (KLSALGLNAV) that is more common in HCV isolates collected in Asia but is rare in sequences from Europe and North America. The superior immunogenicity and cross-reactivity of this relatively rare epitope variant were confirmed by using HCV-specific memory CD8+ T cells from people who inject drugs, who are frequently exposed to HCV. Collectively, the data suggest that sequence differences at the epitope level between HCV isolates substantially impact CD8+ T cell priming and the degree of cross-reactivity with other epitope variants. IMPORTANCE The results have important implications for vaccine design against highly variable pathogens and suggest that evidence-based selection of the vaccine antigen sequence may improve immunogenicity and T cell cross-reactivity. Cross-reactive CD8+ T cells are likely beneficial for immune control of transmitted viruses carrying epitope variants and for prevention of immune escape during acute infection. To this end, rare epitope variants and potentially even altered epitope sequences associated with priming of broadly cross-reactive T cell receptors should be considered for vaccine design and need further testing.

MAGIC biomarkers predict long term outcomes for steroid-resistant acute GVHD

Acute graft-versus-host disease (GVHD) is treated with systemic corticosteroid immunosuppression. Clinical response after 1 week of therapy often guides further treatment decisions, but long-term outcomes vary widely among centers, and more accurate predictive tests are urgently needed. We analyzed clinical data and blood samples taken 1 week after systemic treatment of GVHD from 507 patients from 17 centers of the Mount Sinai Acute GVHD International Consortium (MAGIC), dividing them into a test cohort (n = 236) and 2 validation cohorts separated in time (n = 142 and n = 129). Initial response to systemic steroids correlated with response at 4 weeks, 1-year nonrelapse mortality (NRM), and overall survival (OS). A previously validated algorithm of 2 MAGIC biomarkers (ST2 and REG3α) consistently separated steroid-resistant patients into 2 groups with dramatically different NRM and OS (P < .001 for all 3 cohorts). High biomarker probability, resistance to steroids, and GVHD severity (Minnesota risk) were all significant predictors of NRM in multivariate analysis. A direct comparison of receiver operating characteristic curves showed that the area under the curve for biomarker probability (0.82) was significantly greater than that for steroid response (0.68, P = .004) and for Minnesota risk (0.72, P = .005). In conclusion, MAGIC biomarker probabilities generated after 1 week of systemic treatment of GVHD predict long-term outcomes in steroid-resistant GVHD better than clinical criteria and should prove useful in developing better treatment strategies.

Characterization of a novel OTX2‐driven stem cell program in Group 3 and Group 4 medulloblastoma

Medulloblastoma (MB) is the most common malignant primary pediatric brain cancer. Among the most aggressive subtypes, Group 3 and Group 4 originate from stem/progenitor cells, frequently metastasize, and often display the worst prognosis, yet we know the least about the molecular mechanisms driving their progression. Here, we show that the transcription factor orthodenticle homeobox 2 (OTX2) promotes self‐renewal while inhibiting differentiation in vitro and increases tumor initiation from MB stem/progenitor cells in vivo. To determine how OTX2 contributes to these processes, we employed complementary bioinformatic approaches to characterize the OTX2 regulatory network and identified novel relationships between OTX2 and genes associated with neuronal differentiation and axon guidance signaling in Group 3 and Group 4 MB stem/progenitor cells. In particular, OTX2 levels were negatively correlated with semaphorin (SEMA) signaling, as expression of 9 SEMA pathway genes is upregulated following OTX2 knockdown with some being potential direct OTX2 targets. Importantly, this negative correlation was also observed in patient samples, with lower expression of SEMA4D associated with poor outcome specifically in Group 4 tumors. Functional proof‐of‐principle studies demonstrated that increased levels of select SEMA pathway genes are associated with decreased self‐renewal and growth in vitro and in vivo and that RHO signaling, known to mediate the effects of SEMA genes, is contributing to the OTX2 KD phenotype. Our study provides mechanistic insight into the networks controlled by OTX2 in MB stem/progenitor cells and reveals novel roles for axon guidance genes and their downstream effectors as putative tumor suppressors in MB.

Spontaneous reversion of a lineage switch following an initial blinatumomab-induced ALL-to-AML switch in MLL-rearranged infant ALL

Despite intensified therapy protocols, acute lymphoblastic leukemia (ALL) of infancy remains a difficult-to-treat disease, with a high relapse rate. Only 25% of the patients, treated intensively following the relapse, survive by 3 years. MLL rearrangement is detected in the majority (80%) of patients and is known to be the critical driver for clonal expansion.1,2 Novel immunotherapies spark hope for an effective treatment strategy in the case of chemoresistant disease. Targeting CD19 has been the fundamental principle for both antibody-based immunotherapy (such as blinatumomab) and T-cell-mediated therapy (using chimeric antigen receptor–engineered T cells [CAR T cells]). However, the efficacy of both therapeutic strategies hinges on the maintained surface expression of CD19 as the target molecule. In a fraction of patients, treated with blinatumomab or CAR T cells, downregulation or loss of CD19 expression is observed as one way to escape from immunological pressure.3,4 Other reports,5-11 summarized here (Table 1), describe a lineage switch of the leukemic blasts toward a myeloid phenotype following blinatumomab or CAR T-cell immunotherapy, respectively. Here we report on a child with infant ALL, receiving blinatumomab for early relapse following allogeneic stem cell transplantation (SCT). After only 11 days of treatment, monocytic myeloid blasts displaying an M5 morphology were detected in the peripheral blood and bone marrow, indicating a switch to acute myeloid leukemia (AML). Flow cytometry confirmed a switch of the blast population with an expression of myeloid markers. Most surprisingly, cessation of blinatumomab and a watchful waiting period of 7 days resulted in the spontaneous conversion of leukemia back to the original CD19+ lymphoblastic phenotype. The case documents the high plasticity of these early progenitor blasts, stressing the demand for effective therapy combinations that limit tumor escape. Table 1. Reported cases of a switch in phenotype following immunotherapy