Evolution in health and medicine Sackler colloquium: Making evolutionary biology a basic science for medicine.

New applications of evolutionary biology in medicine are being discovered at an accelerating rate, but few physicians have sufficient educational background to use them fully. This article summarizes suggestions from several groups that have considered how evolutionary biology can be useful in medicine, what physicians should learn about it, and when and how they should learn it. Our general conclusion is that evolutionary biology is a crucial basic science for medicine. In addition to looking at established evolutionary methods and topics, such as population genetics and pathogen evolution, we highlight questions about why natural selection leaves bodies vulnerable to disease. Knowledge about evolution provides physicians with an integrative framework that links otherwise disparate bits of knowledge. It replaces the prevalent view of bodies as machines with a biological view of bodies shaped by evolutionary processes. Like other basic sciences, evolutionary biology needs to be taught both before and during medical school. Most introductory biology courses are insufficient to establish competency in evolutionary biology. Premedical students need evolution courses, possibly ones that emphasize medically relevant aspects. In medical school, evolutionary biology should be taught as one of the basic medical sciences. This will require a course that reviews basic principles and specific medical applications, followed by an integrated presentation of evolutionary aspects that apply to each disease and organ system. Evolutionary biology is not just another topic vying for inclusion in the curriculum; it is an essential foundation for a biological understanding of health and disease.

Adult stem cells as an alternative source of multipotential (pluripotential) cells in regenerative medicine.

Embryonic stem cells are by definition the master cells capable of differentiating into every type of cells either in vitro or in vivo. Several lines of evidence suggest, however, that adult stem cells and even terminally differentiated somatic cells under appropriate microenvironmental cues are able to be reprogrammed and contribute to a much wider spectrum of differentiated progeny than previously anticipated. This has been demonstrated by using tissue- specific stem cells, which like embryonic stem cells do not express CD45 as an exclusive hematopoietic marker (skin, adipose, cord blood and bone marrow- derived stem cells). On the other side, there is a great number of reports which demonstrate that hematopoietic cells (CD45+) from different sources (peripheral blood, cord blood, bone marrow) are also able to cross the tissue boundaries and give rise to the cells of the other germ layers. Herein we discuss the differentiation and reprogramming potential of both hematopoietic and non- hematopoietic stem cells along endodermal, mesodermal and neuroectodermal lineage and their importance for regenerative medicine.

Risk-adjusted therapy of acute lymphoblastic leukemia can decrease treatment burden and improve survival: treatment results of 2169 unselected pediatric and adolescent patients enrolled in the trial ALL-BFM 95.

The trial ALL-BFM 95 for treatment of childhood acute lymphoblastic leukemia was designed to reduce acute and long-term toxicity in selected patient groups with favorable prognosis and to improve outcome in poor-risk groups by treatment intensification. These aims were pursued through a stratification strategy using white blood cell count, age, immunophenotype, treatment response, and unfavorable genetic aberrations providing an excellent discrimination of risk groups. Estimated 6-year event-free survival (6y-pEFS) for all 2169 patients was 79.6% (+/- 0.9%). The large standard-risk (SR) group (35% of patients) achieved an excellent 6y-EFS of 89.5% (+/- 1.1%) despite significant reduction of anthracyclines. In the medium-risk (MR) group (53% of patients), 6y-pEFS was 79.7% (+/- 1.2%); no improvement was accomplished by the randomized use of additional intermediate-dose cytarabine after consolidation. Omission of preventive cranial irradiation in non-T-ALL MR patients was possible without significant reduction of EFS, although the incidence of central nervous system relapses increased. In the high-risk (HR) group (12% of patients), intensification of consolidation/reinduction treatment led to considerable improvement over the previous ALL-BFM trials yielding a 6y-pEFS of 49.2% (+/- 3.2%). Compared without previous trial ALL-BFM 90, consistently favorable results in non-HR patients were achieved with significant treatment reduction in the majority of these patients.

Stem cell transplantation for polycythemia vera.

Polycythemia vera (PV) is a rare disease in children. A 9-year-old male was diagnosed following laboratory results acquired because of an acute appendicitis. Regular phlebotomy was performed for over 2 years followed by alpha-interferon treatment. At the age of 12 years, HLA-matched unrelated stem cell transplantation including T-cell depletion was done. The conditioning regimen consisted of busulfan, cyclophosphamide, and ATG. Chimerism was monitored during the whole post-transplant period. A single dose of donor T-lymphocytes was given at month 3. One year after transplantation, chimerism was complete. The patient is in complete remission and shows no signs of transplant-related morbidity at month 78.

Sequence polymorphism systems for quantitative real-time polymerase chain reaction to characterize hematopoietic chimerism-high informativity and sensitivity as well as excellent reproducibility and precision of measurement.

Sequence polymorphisms (SPs) can serve as genetic markers for quantitative polymerase chain reactions (qPCR) for chimerism analysis, providing a significantly higher sensitivity compared to short tandem repeat PCR. In this study, a panel of 29 selected markers was evaluated in 317 patients with leukemia and myelodysplastic syndrome, who received allogeneic stem cell transplantation. In total, 5415 posttransplantation samples were analyzed. Recipient genotype discrimination was possible in 96% with a mean number of 2.5 (1-7) informative markers per recipient/donor pair. Marker specific standard dilution series from volunteers' DNA served as standard for quantification of chimerism. Sensitivity of the method was < or =1 x 10-3 (0.1% of recipient cells) in 83.3% of the assays. By this method, it was possible to very accurately detect autologous signals in the range from 0% to 0.5% (95% confidence interval [CI] +/-0.2), from 0.5% to 1% (95% CI +/-0.4), from 1% to 2% (95% CI +/-0.6) and from 2% to 5% (95% CI +/-1.2). Reproducibility of the quantified autologous signals was independent from the amount of DNA. This is the first report on a SP-based chimerism system allowing for the performance of chimerism analyses for virtually all patients with high sensitivity, excellent reproducibility, and precision of measurement.

Superiority of allogeneic hematopoietic stem-cell transplantation compared with chemotherapy alone in high-risk childhood T-cell acute lymphoblastic leukemia: results from ALL-BFM 90 and 95.

PURPOSE: The role of hematopoietic stem-cell transplantation (SCT) in first complete remission (CR1) for children with very high-risk (VHR) acute lymphoblastic leukemia (ALL) is still under critical discussion. PATIENTS AND METHODS: In the ALL-Berlin-Frankfurt-Münster (BFM) 90 and ALL-BFM 95 trials, 387 patients were eligible for SCT if there was a matched sibling donor (MSD). T-cell ALL (T-ALL) patients with poor in vivo response to initial treatment represented the largest homogeneous subgroup within VHR patients. RESULTS: Of 191 high-risk (HR) T-ALL patients, 179 patients (94%) achieved CR1. Twenty-three patients received an MSD-SCT. Furthermore, in trial ALL-BFM 95, eight matched unrelated donors (MUDs) and five mismatched family donors (MMFDs) were used. The median time to SCT was 5 months (range, 2.4 to 10.8 months) from diagnosis. The 5-year disease-free survival (DFS) was 67% +/- 8% for 36 patients who received an SCT in CR1 and 42% +/- 5% for the 120 patients treated with chemotherapy alone having an event-free survival time of at least the median time to transplantation (Mantel-Byar, P = .01). Overall survival (OS) rate for the SCT group was 67% +/- 8% at 5 years, whereas patients treated with chemotherapy alone had an OS rate of 47% +/- 5% at 5 years (Mantel-Byar, P = .01). Outcome of patients who received MSD-SCT versus MUD-/MMFD-SCT was comparable (DFS, 65% +/- 10% v 69% +/- 13%, respectively). However, relapses only occurred after MSD-SCT (eight of 23 patients), whereas treatment-related mortality only occurred after MUD-/MMFD-SCT (four of 13 patients). CONCLUSION: SCT in CR1 is superior to treatment with chemotherapy alone for childhood HR-T-ALL.

Pitfalls in detection of contaminating neuroblastoma cells by tyrosine hydroxylase RT-PCR due to catecholamine-producing hematopoietic cells.

BACKGROUND: RT-PCR analysis of compounds of catecholamine metabolism (in particular tyrosine hydroxylase, TH) is widely used for the detection of contaminating neuroblastoma cells in hematopoietic stem cell preparations. Due to reports in the literature showing that hematopoietic cells are also able to produce catecholamines, we investigated whether TH-RT-PCR is really suitable for this purpose. MATERIALS AND METHODS: Besides neuroblastoma cells, mononuclear blood cells, apheresis preparations and hematopoietic stem cells were used for single and nested RT-PCR. In addition to TH, the expressions of dopamine-beta-hydroxylase and noradrenaline transporter were analyzed. RESULTS: Using single RT-PCR, a clear discrimination between neuroblastoma and hematopoietic cells was possible. However, by using nested RT-PCR, the "neuroblastoma markers" were also detected in a significant percentage of non-mobilized mononuclear blood cells, in mononuclear blood cells of healthy donors mobilized with G-CSF, and in highly purified CD34+ and CD133+ stem cells from healthy mobilized donors. CONCLUSION: Our results raise the question of whether the RT-PCR analysis of compounds of catecholamine metabolism is suitable and selective enough to detect the contamination of hematopoietic stem cells by a low number of neuroblastoma cells.

Outcome of allogeneic stem cell transplantation in children with non-malignant diseases.

BACKGROUND AND OBJECTIVES: After allogeneic stem cell transplantation treatment failures are mostly caused by graft rejection or graft-versus-host disease (GVHD). T-cell depletion is an appropriate tool to prevent GvHD. However, it might be associated with an increased risk of graft rejection, which can be recognized by serial and quantitative characterization of chimerism. Thus, pre-emptive immunotherapy might be helpful to avoid graft rejection. DESIGN AND METHODS: We present the outcome of 56 transplants performed in 53 children with non-malignant diseases. T-cell depletion was conducted in 27/56 grafts. When increasing mixed chimerism over 30% autologous cells occurred low dose donor lymphocyte transfusions (DLT) were performed. RESULTS: During the course of the follow-up 29 out of 53 patients achieved complete chimerism or low mixed chimerism (0-1%) and 28/29 remained in continuous complete remission. Donor engraftment failed in 2/53 patients who died of serious infection. Increasing mixed chimerism was found in 19 out of 56 transplantations. Fifteen of these 19 patients received additional immunotherapy with DLT. Eleven out of the 15 remained in complete remission. One of the 15 patients developed GvHD grade III that turned out to extensive chronic GvHD. The 3-year overall survival was 100% for patients transplanted from matched related or unrelated donors and 75% for patients transplanted from mismatched donors. INTERPRETATION AND CONCLUSIONS: We demonstrated that children transplanted for non-malignant diseases have an excellent overall survival. T-cell depletion is associated with an increased risk of graft rejection. Pre-emptive immunotherapy with DLT, administered on the basis of increasing mixed chimerism, is feasible and might prevent graft rejection.

Haematopoietic stem cell transplantation for Shwachman-Diamond disease: a study from the European Group for blood and marrow transplantation.

This report assessed the results of allogeneic stem cell transplantation (allo-SCT) in 26 patients with Shwachman-Diamond disease (SDS) and severe bone marrow abnormalities. The conditioning regimen was based on busulphan (54%), total body irradiation (23%), fludarabine (15%) or other chemotherapy combinations (8%). Standard prevention of graft versus host disease (GVHD) with cyclosporin +/- methotrexate was adopted in 54% of the patients whilst in vivo or in vitro T-cell depletion was used in 17 and four patients respectively. Neutrophil and platelet engraftment were achieved in 21 (81%) and 17 (65%) of 26 patients after a median time of 18 days and 29 days respectively. The incidence of grade III and IV acute GVHD was 24% and of chronic GVHD 29%. Nine patients died after a median time of 70 d, post-SCT. After a median follow-up of 1.1 years, the transplant-related mortality was 35.5% (95% CI 17-54) whilst the overall survival was 64.5% (95% CI 45.7-83.2). Allo-SCT was found to be successful in more than half of SDS patients with severe bone marrow dysfunction. Further improvements would be anticipated by a better definition of the optimum time in the course of disease to transplant and by the adoption of less toxic conditioning regimens.

Effective lysis of lymphoma cells with a stabilised bispecific single-chain Fv antibody against CD19 and FcgammaRIII (CD16).

A recombinant bispecific single-chain fragment variable antibody (bsscFv), directed against the B-cell antigen CD19 and the low affinity Fc-receptor FcgammaRIII (CD16), was designed for use in the treatment of patients with leukaemias and lymphomas. The Fc-portions of whole antibodies were deliberately eliminated in this construct to avoid undesired effector functions. A stabilised bsscFv, ds[CD19 x CD16], was generated, in which disulphide bonds bridging the respective variable light (VL) and variable heavy (VH) chains were introduced into both component single-chain (sc)Fvs. After production in 293T cells and chromatographic purification, ds[CD19 x CD16] specifically and simultaneously bound both antigens. The serum stability of ds[CD19 x CD16] was increased more than threefold when compared with the unstabilised counterpart, while other biological properties were not affected by these mutations. In antibody-dependent cellular cytotoxicity experiments, ds[CD19 x CD16] mediated specific lysis of both CD19-positive malignant human B-lymphoid cell lines and primary tumour cells from patients with B-cell chronic lymphocytic leukaemia or B-cell acute lymphoblastic leukaemia. Natural killer cells, mononuclear cells (MNCs) from healthy donors and, in some instances, MNCs isolated from patients after allogeneic stem cell transplantation, were used as effectors. Thus, ds[CD19 x CD16] holds promise for the treatment of CD19(+) B-lineage malignancies.

Chimaerism analyses and subsequent immunological intervention after stem cell transplantation in patients with juvenile myelomonocytic leukaemia.

Chimaerism analysis was performed by polymerase chain reaction amplification of short-tandem repeat markers in 30 children following haematopoietic stem cell transplantation for juvenile myelomonocytic leukaemia (JMML). Fourteen patients always had complete chimaerism (CC); one of them relapsed after the discontinuation of the study and 13 continued in complete remission (CR). Mixed chimaerism (MC) was noted in 16 patients. Of those 12 patients demonstrated increasing MC (i-MC); 10 relapsed and two achieved CC following discontinuation of immunosuppressive therapy (IST). Four other patients demonstrating only transient MC are alive in CR. MC with up to 20% of autologous cells could be successfully eradicated without induction of severe graft-versus-host disease when IST was reduced or discontinued directly after the first demonstration of MC. At the same time, MC with up to 10% of autologous cells could disappear without intervention. The interval between MC and relapse ranged from 0-320 d (median 38 d). Donor leucocyte infusion was given to six patients with i-MC, but only one patient responded. Peripheral blood seems as valuable as bone marrow for chimaerism studies. In conclusion, serial quantitative chimaerism studies can identify patients with i-MC who are at high risk for relapse of JMML. Immediate withdrawal of IST is advised in these patients.

Sequential expression of adhesion and costimulatory molecules in graft-versus-host disease target organs after murine bone marrow transplantation across minor histocompatibility antigen barriers.

Graft-versus-host disease (GVHD) is a potentially fatal complication after allogeneic bone marrow transplantation. However, few data exist thus far on the molecular signals governing leukocyte trafficking during the disease. We therefore investigated the sequential pattern of distinct adhesion, costimulatory, and apoptosis-related molecules in GVHD organs (ileum, colon, skin, and liver) after transplantation across minor histocompatibility barriers (B10.D2 --> BALB/c, both H-2d). To distinguish changes induced by the conditioning regimen from effects achieved by allogeneic cell transfer, syngeneic transplant recipients (BALB/c --> BALB/c) and irradiated nontransplanted mice were added as controls. Irradiation upregulated the expression of vascular cell adhesion molecule (VCAM)-1, intercellular adhesion molecule (ICAM)-l, and B7-2 in ileum, as well as VCAM-1 and B7-2 in colon, on day 3 in all animals. Whereas in syngeneic mice these effects were reversed from day 9 on, allogeneic recipients showed further upregulation of VCAM-1, ICAM-1, B7-1, and B7-2 in these organs on day 22, when GVHD became clinically evident. Infiltration of CD4+ and CD8+ donor T cells was noted on day 9 in skin and liver and on day 22 in ileum and colon. Surprisingly, the expression of several other adhesion molecules, such as ICAM-2, platelet-endothelial cell adhesion molecule 1, E-selectin, and mucosal addressin cell adhesion molecule 1, did not change. Proapoptotic and antiapoptotic markers were balanced in GVHD organs with the exception of spleen, in which a preferential expression of the proapoptotic Bax could be noted. Our results indicate that irradiation-induced upregulation of VCAM-1, ICAM-1, and B7-2 provides early costimulatory signals to incoming donor T cells in the intestine, followed by a cascade of proinflammatory signals in other organs once the alloresponse is established.

Concurrent detection of minimal residual disease (MRD) in childhood acute lymphoblastic leukaemia by flow cytometry and real-time PCR.

Minimal (i.e. submicroscopic) residual disease (MRD) predicts outcome in childhood acute lymphoblastic leukaemia (ALL). To be used clinically, MRD assays must be reliable and accurate. Two well-established techniques, flow cytometry (FC) and polymerase chain reaction (PCR), can detect leukaemic cells with a sensitivity of 0.01% (10(-4)). We analysed diagnostic samples of 45 ALL-patients (37 B-lineage ALL, eight T-lineage ALL) by four-colour FC and real-time PCR. Leukaemia-associated immunophenotypes, at a sensitivity of MRD detection by FC at the 0.01% level, were identified in 41 cases (91%); antigen-receptor gene rearrangements suitable for MRD detection with a sensitivity of 0.01% or better by PCR were identified in 38 cases (84%). The combined use of FC and PCR allowed MRD monitoring in all 45 patients. In 105 follow-up samples, MRD estimates by both methods were highly concordant, with a deviation factor of <5 by Bland-Altman analysis. Importantly, the concordance between FC and PCR was also observed in regenerating bone marrow samples containing high proportions of CD19(+) cells, and in samples studied 24 h after collection. We conclude that both MRD assays yield generally concordant results. Their combined use should enable MRD monitoring in virtually all patients and prevent false-negative results due to clonal evolution or phenotypic shifts.

Onset of thymic recovery and plateau of thymic output are differentially regulated after stem cell transplantation in children.

Thymus-dependent T-cell regeneration is a major pathway for immune reconstitution after stem cell transplantation in children. Therefore, we prospectively assessed T-cell dynamics and thymic function in 164 pediatric patients between 1 and 124 months after transplantation by measuring T-cell receptor recombination excision circles and spontaneous expression of Ki67 in peripheral T-cell subsets. We analyzed the effect of recipient age, conditioning regimen, type of donor and graft, stem cell dose, and graft-versus-host disease on the onset and the plateau of thymic output. A high rate of spontaneous proliferation in early-reconstituting naive and memory T cells inversely correlated with total T-cell numbers. Accordingly, T-cell receptor recombination excision circle content was diminished in early-appearing naive T cells. A multivariate analysis revealed that the onset of thymic recovery was inversely correlated only with recipient age ( P < .0002), whereas the plateau of thymic output was higher in patients receiving increased stem cell numbers ( P < .0022). Donor type, stem cell source, and conditioning regimen influenced none of the analyzed parameters. In conclusion, lymphopenia-driven proliferation is important for T-cell homeostasis in children early after stem cell transplantation, but it might result in underestimation of thymic function. Onset and plateau of thymic activity are independently regulated by different transplant-related factors.

Detection of adenovirus-specific T cells in children with adenovirus infection after allogeneic stem cell transplantation.

Human adenovirus (ADV) infection is an important complication post allogeneic stem cell transplantation (SCT), especially in children, with significant morbidity and mortality despite new antiviral treatment strategies. Although the control of infection seems to require T cells, characterization of ADV-specific T cells post-SCT has not been reported to date. Therefore, we prospectively studied the occurrence of ADV-specific T cells in children with (n = 21) and without (n = 25) ADV-infection postallogeneic SCT and in healthy donors (n = 53). ADV-associated mortality occurred in seven of 21 children. After stimulation ex vivo with ADV-lysate, interferon-gamma-secreting T cells were analysed by flow cytometry. All the patients with ADV-associated mortality had no specific T cells, although reconstitution of absolute lymphocyte counts exceeded 0.3 x 10(9)/l within 30 d post-transplant. Patients who cleared ADV infection had significantly higher frequencies (mean +/- standard deviation, 0.56 +/- 0.5%) of ADV-specific T cells until day 200 post-SCT, than patients without ADV-infection (0.12 +/- 0.1%). These data suggest that ADV-specific T-cell reconstitution is protective against life-threatening ADV-infections postallogeneic SCT.

Children with myelodysplastic syndrome (MDS) and increasing mixed chimaerism after allogeneic stem cell transplantation have a poor outcome which can be improved by pre-emptive immunotherapy.

We recently reported that virtually all children with acute leukaemia and myelodysplastic syndrome (MDS) who develop the phenotype of increasing mixed chimaerism (MC) after allogeneic stem cell transplantation (allo-SCT) will relapse. We therefore performed a prospective, multi-centre study focused on children with MDS (n = 65; advanced MDS = 44, refractory cytopenia = 21) after allo-SCT in order to determine to what extent relapse can be prevented by pre-emptive immunotherapy on the basis of increasing MC. Analyses of chimaerism in 44 patients with advanced MDS revealed 31 cases with complete chimaerism (CC)/low-level MC/transient MC, 11 cases with increasing MC and two cases with decreasing MC. The same analyses in 21 MDS patients with refractory cytopenia revealed 17 cases with CC/low-level MC, one case with increasing MC and three cases with decreasing MC. Pre-emptive immunotherapy performed on each patient that showed increasing MC improved event-free survival from 0%, as seen in prior studies, to 50%. We therefore conclude that pre-emptive immunotherapy is an effective treatment option to prevent impending relapse in children with MDS after allo-SCT.

Cytotoxic minor histocompatibility antigen HA-1-specific CD8+ effector memory T cells: artificial APCs pave the way for clinical application by potent primary in vitro induction.

Induction of cytotoxic T lymphocytes (CTLs) for treatment of relapsed leukemia after allogeneic stem-cell transplantation is hindered by the laborious and time-consuming procedure of generating dendritic cells for antigen presentation. Artificial antigen-presenting cells (aAPCs) offer the advantage of being readily available in sufficient numbers, thus allowing for a highly standardized in vitro induction of CTLs. We generated aAPCs coated with anti-CD28 antibody (Ab) and either high-density (HD) or low-density (LD) major histocompatibility complex (MHC) class I molecules loaded with HA-1(H), a nonapeptide derived from the hematopoiesis-restricted minor histocompatibility antigen HA-1. HD- and LD-aAPCs potently induced HA-1(H)-specific CD8+ CTLs from untouched CD8+ T cells of healthy donors. CTLs were subsequently purified by magnetic-activated cell sorting. HD- as well as LD-aAPC-induced CTLs exerted high HA-1H-specific cytotoxicity, resembled T(c)1 effector memory cells, survived a long time in vitro, and were expanded by a factor varying between 8.2 x 10(4) and 51 x 10(4). The T-cell receptor (TCR) repertoire of HA-1H tetramer-positive CTLs was oligoclonal with a prominent usage of Vbeta6. The TCR repertoire of tetramer-positive CTLs was distinct from and more restricted than that of tetramer-negative cells. These findings indicate that aAPCs are attractive tools for the ex vivo generation of HA-1H-specific CTLs suitable for immunotherapy of relapsed leukemia.