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Recently, a rare type of relapse was reported upon treating a B cell acute lymphoblastic leukemia (B-ALL) patient with anti-CD19 chimeric antigen receptor (CAR)-T cells caused by unintentional transduction of residual malignant B cells (CAR-B cells). We show that anti-CD19 and anti-CD20 CARs are presented on the surface of lentiviral vectors (LVs), inducing specific binding to the respective antigen. Binding of anti-CD19 CAR-encoding LVs containing supernatant was reduced by CD19-specific blocking antibodies in a dose-dependent manner, and binding was absent for unspecific LV containing supernatant. This suggests that LVs bind via displayed CAR molecules to CAR antigen-expressing cells. The relevance for CAR-T cell manufacturing was evaluated when PBMCs and B-ALL malignant B cells were mixed and transduced with anti-CD19 or anti-CD20 CAR-displaying LVs in clinically relevant doses to mimic transduction conditions of unpurified patient leukapheresis samples. Malignant B cells were transduced at higher levels with LVs displaying anti-CD19 CARs compared to LVs displaying non-binding control constructs. Stability of gene transfer was confirmed by applying a potent LV inhibitor and long-term cultures for 10 days. Our findings provide a potential explanation for the emergence of CAR-B cells pointing to safer manufacturing procedures with reduced risk of this rare type of relapse in the future., Graphical abstract, Schaser and colleagues found that lentiviral vectors encoding B cell-specific CARs display the respective CAR on the particle surface. The LV-displayed CARs retained their antigen-binding potential and specifically promoted transduction of malignant B cells. These findings provide a potential explanation for the emergence of CAR-expressing malignant cells in B-ALL patients.

A major roadblock prohibiting effective cellular immunotherapy of pancreatic ductal adenocarcinoma (PDAC) is the lack of suitable tumor-specific antigens. To address this challenge, here we combine flow cytometry screenings, bioinformatic expression analyses and a cyclic immunofluorescence platform. We identify CLA, CD66c, CD318 and TSPAN8 as target candidates among 371 antigens and generate 32 CARs specific for these molecules. CAR T cell activity is evaluated in vitro based on target cell lysis, T cell activation and cytokine release. Promising constructs are evaluated in vivo. CAR T cells specific for CD66c, CD318 and TSPAN8 demonstrate efficacies ranging from stabilized disease to complete tumor eradication with CD318 followed by TSPAN8 being the most promising candidates for clinical translation based on functionality and predicted safety profiles. This study reveals potential target candidates for CAR T cell based immunotherapy of PDAC together with a functional set of CAR constructs specific for these molecules., There is an unmet clinical need to identify therapeutic options for the treatment of pancreatic cancer (PDAC). Here the authors present a systematic screening approach for the identification of potential PDAC cell surface target candidates for CAR-T cell based immunotherapy, followed by their functional validation in preclinical models.

A domain that is often neglected in the assessment of chimeric antigen receptor (CAR) functionality is the extracellular spacer module. However, several studies have elucidated that membrane proximal epitopes are best targeted through CARs comprising long spacers, while short spacer CARs exhibit highest activity on distal epitopes. This finding can be explained by the requirement to have an optimal distance between the effector T cell and target cell. Commonly used long spacer domains are the CH2-CH3 domains of IgG molecules. However, CARs containing these spacers generally show inferior in vivo efficacy in mouse models compared to their observed in vitro activity, which is linked to unspecific Fcγ-Receptor binding and can be abolished by mutating the respective regions. Here, we first assessed a CAR therapy targeting membrane proximal CD20 using such a modified long IgG1 spacer. However, despite these mutations, this construct failed to unfold its observed in vitro cytotoxic potential in an in vivo model, while a shorter but less structured CD8α spacer CAR showed complete tumor clearance. Given the shortage of well-described long spacer domains with a favorable functionality profile, we designed a novel class of CAR spacers with similar attributes to IgG spacers but without unspecific off-target binding, derived from the Sialic acid-binding immunoglobulin-type lectins (Siglecs). Of five constructs tested, a Siglec-4 derived spacer showed highest cytotoxic potential and similar performance to a CD8α spacer in a CD20 specific CAR setting. In a pancreatic ductal adenocarcinoma model, a Siglec-4 spacer CAR targeting a membrane proximal (TSPAN8) epitope was efficiently engaged in vitro, while a membrane distal (CD66c) epitope did not activate the T cell. Transfer of the TSPAN8 specific Siglec-4 spacer CAR to an in vivo setting maintained the excellent tumor killing characteristics being indistinguishable from a TSPAN8 CD8α spacer CAR while outperforming an IgG4 long spacer CAR and, at the same time, showing an advantageous central memory CAR T cell phenotype with lower release of inflammatory cytokines. In summary, we developed a novel spacer that combines cytotoxic potential with an advantageous T cell and cytokine release phenotype, which make this an interesting candidate for future clinical applications.

ETV6-RUNX1 is associated with the most common subtype of childhood leukemia. As few ETV6-RUNX1 carriers develop precursor B-cell acute lymphocytic leukemia (pB-ALL), the underlying genetic basis for development of full-blown leukemia remains to be identified, but the appearance of leukemia cases in time-space clusters keeps infection as a potential causal factor. Here, we present in vivo genetic evidence mechanistically connecting preleukemic ETV6-RUNX1 expression in hematopoetic stem cells/precursor cells (HSC/PC) and postnatal infections for human-like pB-ALL. In our model, ETV6-RUNX1 conferred a low risk of developing pB-ALL after exposure to common pathogens, corroborating the low incidence observed in humans. Murine preleukemic ETV6-RUNX1 pro/preB cells showed high Rag1/2 expression, known for human ETV6-RUNX1 pB-ALL. Murine and human ETV6-RUNX1 pB-ALL revealed recurrent genomic alterations, with a relevant proportion affecting genes of the lysine demethylase (KDM) family. KDM5C loss of function resulted in increased levels of H3K4me3, which coprecipitated with RAG2 in a human cell line model, laying the molecular basis for recombination activity. We conclude that alterations of KDM family members represent a disease-driving mechanism and an explanation for RAG off-target cleavage observed in humans. Our results explain the genetic basis for clonal evolution of an ETV6-RUNX1 preleukemic clone to pB-ALL after infection exposure and offer the possibility of novel therapeutic approaches., J. Hauer has been supported by the German Children's Cancer Foundation, DJCLS 02R/2016, KKS A2016/07 and from the “Forschungskommission” of the medical faculty of the Heinrich Heine University. M. Müschen is an HHMI Faculty Scholar (HHMI-55108547) and supported by NIH/NCI through an Outstanding Investigator Award (R35CA197628) to M. Müschen, a Wellcome Trust Senior Investigator Award, the Leukemia and Lymphoma Society, the California Institute for Regenerative Medicine, the William Lawrence and Blanche Hughes Foundation. A. Borkhardt has been supported by the German Children's Cancer Foundation and the Federal Ministry of Education and Research, Bonn, Germany. Research in I. Sánchez-García's group is partially supported by FEDER and by MINECO (SAF2012-32810, SAF2015-64420-R and Red de Excelencia Consolider OncoBIOSAF2014-57791-REDC), Instituto de Salud Carlos III (PIE14/00066), ISCIII- Plan de Ayudas IBSAL 2015 Proyectos Integrados (IBY15/00003), by Junta de Castilla y León (BIO/SA51/15, CSI001U14, UIC-017, and CSI001U16), Fundacion Inocente Inocente and by the ARIMMORA project (European Union's Seventh Framework Programme (FP7/2007-2013) under grant agreement no. 282891). I. Sánchez-García's lab is a member of the EuroSyStem and the DECIDE Network funded by the European Union under the FP7 program. A. Borkhardt and I. Sánchez-García have been supported by the German Carreras Foundation (DJCLS R13/26). Research in C. Vicente-Dueñas' group is partially supported by a “Miguel Servet” grant (CP14/00082-AES 2013-2016-FEDER) from the Instituto de Salud Carlos III (Ministerio de Economía y Competitividad) and by the Lady Tata International Award for Research in Leukaemia 2016–2017. A. Martín-Lorenzo and G. Rodríguez-Hernández were supported by FSE-Conserjería de Educación de la Junta de Castilla y León (CSI001-13 and CSI001-15, respectively).

Seen from a historical perspective the health and illness of a physician is not at all his private matter; they have an impact on his relationship with his patients and the public. Drawing on sources from Graeco-Roman times up to now information referring to this subject is scarce and heterogeneous. However, it is possible to differentiate between three main concepts encompassing various periods: In those reaching approximately until 1700 the physician's personal health and recovery were his credentials of his art of healing. In the following 250 years he increasingly stylized himself as a victim in the service of his patients and science. After 1970 helpless helpers are threatened by mental illness.

Purpose: Metformin (MF) acts as a tumour-suppressor in renal cell carcinoma (RCC) by inhibiting the AKT/mTOR pathway via AMPK activation. Here, we explore the influence of miR-21 and its target gene PTEN on MF effects in CAKI-1 and CAKI-2 cells. Methods: Proliferation assays (MTS) and qRT-PCR after transient transfection with pre- and anti-miR-21 and MF treatment were conducted. AMPK-dependency was assessed via transfection of siAMPK. The expression of PTEN, AKT and miR-21 after transient pre-miR-21 transfection and MF treatment was analysed. Results: We demonstrate that CAKI-1 cells, which were found to be less sensitive towards MF, showed a significant higher miR-21 and lower PTEN expression than CAKI-2. This was confirmed in a primary RCC collective (n = 28): miR-21 and PTEN expression correlated negatively. MF treatment lowered miR-21 AMPK-dependently and increased PTEN expression in the cell lines. Ectopic miR-21 regulation modulated MF sensitivity. Western blot analysis showed that pre-miR-21 transfection and MF treatment regulated PTEN expression with impact on pAKT levels in the cells. Conclusions: We show that differing MF sensitivity in RCC cells is associated with and mediated through the regulation of miR-21/PTEN expression with an impact on subsequent AKT signalling. This provides imaginable clinical implications regarding MF therapy of RCC patients for the future.

ETV6-RUNX1 is associated with the most common subtype of childhood leukemia. Pre-leukaemic clones carrying ETV6-RUNX1 oncogenic lesions are frequently found in neonatal cord blood, but only few ETV6-RUNX1 carriers develop pB-ALL. The highly demanding and pending challenge is to reveal the multistep natural history of ETV6-RUNX1 pB-ALL, because it can offer non-toxic prophylactic interventions to preleukemic carriers. However, the lack of a genetically engineered ETV6-RUNX1 mouse model mimicking the human pB-ALL has hampered our understanding of the pathogenesis of this disease. This rule has now been broken in a study of the effect of the ETV6-RUNX1 oncogene in cancer development in a mouse model in which oncogene expression is restricted to the stem cell compartment. In this article, we review the different attempts to model this disease, including the recent representative success stories and we discuss its potential application to both identify etiologic factors of childhood ETV6-RUNX1 pB-ALL and prevent the conversion of a preleukemic clone in an irreversible transformed state., J.H. has been supported by the German Children’s Cancer Foundation, DJCLS 02R/2016, KKS A2016/07 and from the “Forschungskommission” of the medical faculty of the Heinrich Heine University. A.B. has been supported by the German Children’s Cancer Foundation and the Federal Ministry of Education and Research, Bonn, Germany. Funding to S.N.C. from Salus Sanguinis, Fondation contre le cancer Belgium; Interuniversity Attraction Poles IAP and ARC 10/15-027 is acknowledged. Research in ISG group is partially supported by FEDER and by MINECO (SAF2012-32810, SAF2015-64420-R and Red de Excelencia Consolider OncoBIO SAF2014-57791-REDC), Instituto de Salud Carlos III (PIE14/00066), ISCIII- Plan de Ayudas IBSAL 2015 Proyectos Integrados (IBY15/00003), by Junta de Castilla y León (BIO/SA51/15, CSI001U14, UIC017, and CSI001U16), and by the Fundacion Inocente Inocente. ISG lab is a member of the EuroSyStem and the DECIDE Network funded by the European Union under the FP7 program. A.B. and ISG have been supported by the German Carreras Foundation (DJCLS R13/26). Research in CVD group is partially supported by a “Miguel Servet” Grant (CP14/00082 - AES 2013-2016 - FEDER) from the Instituto de Salud Carlos III (Ministerio de Economía y Competitividad) and by the Lady Tata International Award for Research in Leukaemia 2016–2017.

The nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 (NF-κB1) is a master regulator of immune and inflammatory responses.[1][1],[2][2] NF-κB1 belongs to the NF-κB/Rel family of transcription factors that consists of five members in humans: NF-κB1 (p105/p50), NF-κB2 (p100/p52

Background: Bednar’s aphthae can still be found in newborn infants but have nearly disappeared from recent paediatric literature. Objectives: To find out the incidence of Bednar’s aphthae among newborn infants during the first week of life and the factors associated with the ulcers. Methods: Clinical findings and statistical data were documented of 1,654 infants routinely examined for preventive reasons in two obstetric hospitals in Cologne, Germany. Results: Bednar’s aphthae were found in 236 of the 1,494 neonates examined (15.8%) in whom the whole palate could be visualized. They were associated with spontaneous birth at term, nutrition with formula, and mucosal hyperaemia of the typical anatomical location. Conclusions: Bednar’s aphthae are not a rare phenomenon and still commonly occur in neonates.

The construction of new mediator-free and fully electroactive protein multilayer electrodes based on DNA and cyt c is demonstrated. The assembly is characterized by a rather high stability, effective electron transfer, and a tunability of the electroactive protein amount by the number of deposited cyt c layers. Keywords: Layer-by-layer assembly, Multilayer electrode, DNA, Cyt c electrochemistry

Background The methicillin-resistant Staphylococcus aureus (MRSA) surface protein Pls (plasmin sensitive) reduces adhesion to host proteins and cellular invasiveness by an unknown mechanism that requires Pls expression. Here, we tested the effect of Pls expression using different pls-negative backgrounds. Methods Three pls-negative strains (the methicillin-susceptible Staphylococcus aureus strains Cowan I and 6850 and the MRSA strain ST239-635/93, which harbors staphylococcal cassette chromosome [SCC] mec type III) were transformed. Constructs used were full-length pls (pPLS4), pls-DeltaLPDTG (no sortase motif; pPLS5), and pls-DeltaSD (no serine-aspartic acid [SD] repeats; pPLS6). Adherence, invasiveness, gene expression, and surface expression were quantified by photometry, flow cytometry, real-time reverse-transcription polymerase chain reaction, and a modified enzyme-linked immunosorbent assay, respectively. Results In Pls-expressing strains (those with pPLS4), adherence to immobilized fibronectin (Fn) and binding of soluble Fn was reduced by approximately 20% and approximately 25%, respectively. Invasion of 293 cells and EA.hy 926 cells was reduced by up to 85%. Surprisingly, transcription of fnbA and spa was up-regulated, but transcription of clfA and hla was down-regulated. Pls and Fn-binding protein (FnBP) surface expression was increased. Competition with purified FnBPA, but not with Pls, reduced invasiveness by approximately 90%. The invasiveness of 6850 (pPLS5) and of 6850 (pPLS6) was reduced by only approximately 20% and approximately 15%, respectively. Conclusion Expression of cell wall-anchored Pls reduces adherence and invasiveness independently of the MRSA/SCCmec background. This occurs despite early up-regulation of fnbA transcription and FnBP surface expression. Thus, Pls acts by steric hindrance rather than another mechanism.

Literary and medical writers have written about elderly people—their health, constitution, and treatment—since the dawn of history. The dramatic demographic changes that have taken place within industrial nations in recent times have certainly imparted this theme with a heretofore-unknown explosiveness. Joannes Stobaios' (5th c.) three chapters “On old age” have long been largely unappreciated in the history of gerontology and geriatrics. This late Ancient author collected numerous citations from other, earlier authors who lived between 800 BCE and 400 CE and brought them together thematically in an anthology of monumental scope. The work contains the only versions—albeit sometimes fragmentary—of many texts now otherwise lost. This is also true for the gerontological writings; Stobaios proved that, as early as the end of the Ancient period, an appreciable corpus of relevant texts existed, of which only very little—excepting Cicero's well-known text “On old age” (Cato maior de senectute)—survived into the modern period. These texts reveal that the question as to whether old age is a burden or a joy is ancient and evidently insoluble. This article presents the gerontological aspects of Stobaios' work and places them in their historical context. This will demonstrate that Stobaios was also a defender of seniority who made clear the value, as well as the tasks and ethical duties, of this phase of life. From the Ancients' perspective, this was not a medical but a moral obligation.

Cognitive impairment in old age is one of the most important topics in modern geriatrics. This article discusses the historical dimensions of this phenomenon. To this end, a number of primary sources ranging from antiquity to the modern era are evaluated. Although a physiology and pathology of old age were conceptualized in Greco-Roman times, cognitive impairment in old age remained a marginal issue until the 17th century. Alternatively, after 1500, medicine boasted detailed theories on the physiology and pathology of old age. There are several possible explanations for this unusual situation. Underlying conflict between idealistic and materialistic views of man played a decisive role, for these concepts differed considerably regarding the intellectual and mental functioning of the soul as well as the effects of the passage of time. After Cartesianism and Iatromechanism had pushed these traditional boundaries back, the problem of cognitive impairment in old age was increasingly regarded as a physical illness and began to receive more attention. Just as its philosophical and theological context shaped early modern medicine, contemporary nonmedical disciplines such as genetics, (neuro-)biology, and the information sciences influence modern research.

Earlier in the past century, infections were regarded as the most likely cause of childhood B-cell precursor acute lymphoblastic leukemia (pB-ALL). However, there is a lack of relevant biologic evidence supporting this hypothesis. We present in vivo genetic evidence mechanistically connecting inherited susceptibility to pB-ALL and postnatal infections by showing that pB-ALL was initiated in Pax5 heterozygous mice only when they were exposed to common pathogens. Strikingly, these murine pB-ALLs closely resemble the human disease. Tumor exome sequencing revealed activating somatic, nonsynonymous mutations of Jak3 as a second hit. Transplantation experiments and deep sequencing suggest that inactivating mutations in Pax5 promote leukemogenesis by creating an aberrant progenitor compartment that is susceptible to malignant transformation through accumulation of secondary Jak3 mutations. Thus, treatment of Pax5 leukemic cells with specific JAK1/3 inhibitors resulted in increased apoptosis. These results uncover the causal role of infection in pB-ALL development. [Significance]: These results demonstrate that delayed infection exposure is a causal factor in pB-ALL. Therefore, these findings have critical implications for the understanding of the pathogenesis of leukemia and for the development of novel therapies for this disease., J. Hauer has been supported by the German Children’s Cancer Foundation and from the “Forschungskommission” of the medical faculty of the Heinrich Heine University and the “Strategischer Forschungsfond” of the Heinrich Heine University. S. Ginzel has been supported by a scholarship from the Hochschule Bonn-Rhein-Sieg. A. Borkhardt has been supported by the German Children’s Cancer Foundation and the Federal Ministry of Education and Research, Bonn, Germany. Funding to S.N. Constantinescu from Salus Sanguinis, Fondation contre le cancer Belgium; Interuniversity Attraction Poles IAP, and ARC 10/15-027 is acknowledged. Research in the I. Sánchez-García group is partially supported by FEDER and MINECO (SAF2012-32810 and Red de Excelencia Consolider OncoBIO SAF2014-57791-REDC), Instituto de Salud Carlos III (PIE14/00066), Junta de Castilla y León (BIO/SA32/14 , BIO/SA51/15, and CSI001U14), Fundacion Inocente Inocente, and the ARIMMORA project [European Union’s Seventh Framework Programme (FP7/2007- 2013) under grant agreement no. 282891]. The I. Sánchez-García lab is a member of the EuroSyStem and the DECIDE Network funded by the European Union under the FP7 program. A. Borkhardt and I. SánchezGarcía have been supported by the German Carreras Foundation (DJCLS R13/26). Research in the C. Vicente-Dueñas group is partially supported by a “Miguel Servet” Grant (CP14/00082 - AES 2013-2016) from the Instituto de Salud Carlos III (Ministerio de Economía y Competitividad). Research at C. Cobaleda’s lab was partially supported by FEDER, Fondo de Investigaciones Sanitarias (PI13/00160 and PI14/00025), and from an institutional grant from the Fundacion Ramon Areces. A. MartínLorenzo was supported by FSE-Conserjería de Educación de la Junta de Castilla y León (CSI001-13).

Staphylococcus aureusis a major cause of a variety of both local and systemic infections. It can invade human host cells, a process that may account for disseminated and recurrent infections.S. aureuspostinvasion events in nonprofessional phagocytes are only partially understood. While morphological data suggest a phagosomal escape, there is a lack of corroborating functional data. Using a combination of pH determination and morphological techniques, we have tested the integrity ofStaphylococcus-containing phagosomes in 293 (HEK-293), HeLa, and EA.hy926 cells over time. Rapid acidification ofS. aureus-containing phagosomes occurred and was sustained for up to 24 h. AllS. aureusstrains tested displayed equally sustained intraphagosomal pH levels without exhibiting any correlation with pH level and hemolytic activity. The membrane morphology of the phagosomal compartment was heterogeneous, even under conditions where acidic pH was fully maintained, an observation incompatible with phagolysosomal membrane destruction. As an exception,S. aureusstrain 6850 showed a reduced phagosomal acidification signal 6 h after invasion. Additionally, only strain 6850 failed to localize to LAMP-1-positive vesicles in HeLa cells, although this was observed only rarely. Several other strongly beta-hemolytic strains did not modulate phagolysosomal pH, suggesting thatS. aureusα-toxin and β-toxin are not sufficient for this process. Taken together, our data suggest thatS. aureus-containing phagolysosomes generally remain functionally intact in nonprofessional phagocytes, thereby contrasting with transmission electron micrographic results.

Staphylococcus aureus reacts to changing environmental conditions such as heat, pH, and chemicals through global regulators such as the sae ( S. aureus exoprotein expression) two-component signaling system. Subinhibitory concentrations of some antibiotics were shown to increase virulence factor expression. Here, we investigated the S. aureus stress response to sublethal concentrations of a commonly used biocide (Perform), by real-time quantitative PCR (qRT-PCR), promoter activity assay, sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and a flow cytometric invasion assay. Perform, acting through the production of reactive oxygen species, generally downregulated expression of extracellular proteins in strains 6850, COL, ISP479C but upregulated these proteins in strain Newman. Upregulated proteins were sae dependent. The Perform component SDS, but not paraquat (another oxygen donor), mimicked the biocide effect. Eap (extracellular adherence protein) was most prominently augmented. Upregulation of eap and sae was confirmed by qRT-PCR. Promoter activity of sae P1 was increased by Perform and SDS. Both substances enhanced cellular invasiveness, by 2.5-fold and 3.2-fold, respectively. Increased invasiveness was dependent on Eap and the sae system, whereas agr , sarA , sigB , and fibronectin-binding proteins had no major effect in strain Newman. This unique response pattern was due to a point mutation in SaeS (the sensor histidine kinase), as demonstrated by allele swapping. Newman saePQRS ISP479C behaved like ISP479C, whereas saePQRS Newman rendered ISP479C equally responsive as Newman. Taken together, the findings indicate that a point mutation in SaeS of strain Newman was responsible for increased expression of Eap upon exposure to sublethal Perform and SDS concentrations, leading to increased Eap-dependent cellular invasiveness. This may be important for understanding the regulation of virulence in S. aureus .

Background: The link between longevity and diet is of great interest to biological and gerontological research. The fact that relevant knowledge has generally been available for many centuries is often not remarked upon. Objective: This article examines three aspects of early modern Western medicine which thematize the following links between the elderly, longevity and caloric intake: (1) the question of a diet specifically tailored to old age as background to certain theories of aging; (2) the transfer of these dietetic concepts to younger patients in order to improve health and extend life, and (3) the promotion of dieting in order to avoid the consequences of plethora and to retard the aging process. Methods: A number of Latin texts from premodern medical and health literature will be examined and their contents will be analyzed for material relating to diet for the elderly and longevity in their historic contexts. Results: We will clearly indicate fundamental parallels as well as differences between historic and modern scientific thought. We will thereby show that although a modern understanding of hormones and molecular genetics was obviously lacking, basic knowledge of the influence of nutrition on old age was prevalent. In contrast, the early modern lay concept of longevity through calorie reduction was based on coincidental observation. Conclusion: These premodern, but nonetheless rational ideas must be integrated into the socio-cultural setting and the question must be raised as to the link between contemporary research aims and social reality.

Pediatric acute lymphoblastic leukemia (ALL) is characterized by recurrent chromosomal translocations that frequently occur in utero in preleukemic cells. The translocation t(12;21) resulting in the formation of the chimeric transcription factor ETV6-RUNX1 is the most frequent structural aberration occurring in 25% of B-cell ALL. A previous study suggested that ETV6-RUNX1 positive preleukemic cells are present in every hundredth human newborn, thus exceeding the actually observed incidence of ETV6-RUNX1 positive ALL in children by a factor of 100. This finding indicated that secondary, but relatively rare cooperating oncogenic hits are necessary for the development of overt leukemia. However, later studies could not confirm this high incidence of preleukemic cells in newborns. To analyze the actual frequency of ETV6-RUNX1 preleukemic cells in newborns we developed a PCR based method termed genomic inverse PCR for exploration of ligated breakpoints (GIPFEL) and applied this technique to a population-based screening of ≈1000 cord blood samples from healthy newborns. The GIPFEL method is capable to detect the most common gene fusions associated with childhood leukemia without prior knowledge of the exact breakpoint. In the case of ETV6-RUNX1 positive leukemia, GIPFEL exploits the unique presence of a genomic fragment joining material from chromosomes 12 and 21 in the translocation-positive cells. These fragments can be digested and re-circularized by ligation creating a junction across the restriction site whose sequence can be predicted from published genome data. Importantly, the ligation site is independent of the translocation point within the individual DNA circle. The published breakpoint regions of the ETV6 and RUNX1 genes involved in the translocation were analyzed in silico for restriction sites that allow digestion of all possible translocation events to yield fragments smaller than approximately 50 kb. This condition was met for ETV6-RUNX1 breakpoints by digestion with SacI. Primer pairs were designed amplifying the complete set of theoretically predicted circularized fragments requiring 36 primers for the ETV6-RUNX1 translocation. Genomic DNA was prepared from cell lines, diagnostic specimens from ALL patients, peripheral blood from healthy donors and cord blood samples from newborns by column purification. The equivalent of approximately 4x105 cells (2.5 µg DNA) was subjected to the SacI restriction digest, ligated and remaining linear DNA was removed by exonuclease III. After ethanol precipitation the reaction products were subjected to a partially multiplexed, semi-nested PCR to quantify all possible ligation/junction products specific for the translocation. An internal RUNX1 genomic ligation product served as a quality control and allowed the relative quantification of the translocation product. In a first proof-of-principle study employing the ETV6-RUNX1 translocation positive cell line REH, process optimization close to the theoretical limits was carried out. Cell dilution and mixing studies revealed that under optimal conditions approximately 40 translocation positive cells (=10-4) present in the input DNA are sufficient to produce a reliable output signal. The method was next tested in a blinded study with 60 samples obtained from ETV6-RUNX1 diagnostic ALL samples. ETV6-RUNX1 samples positive at 10-4, being diluted from these diagnostic samples, still gave a reliable output signal. There was no false positive result. Detection coverage (=sensitivity) was 64%. This method was then applied to a retrospective sample set of cryopreserved anonymized cord blood samples of ≈1000 healthy newborns to determine frequency and levels of translocation-positive cells. First results will be presented. In conclusion this population-based study will allow an estimate of the actual incidence of ETV6-RUNX1 positive preleukemic cells in healthy newborns. The results will enable us to evaluate the penetrance and leukemia inducing potential of the chimeric transcription factor ETV6-RUNX1 in human newborns and will provide a basis for the assessment of potential secondary environmental or spontaneously occurring cooperating oncogenic lesions in ETV6-RUNX1 positive childhood leukemia. Disclosures No relevant conflicts of interest to declare.

Un changement radical est intervenu dans la pensee medicale du XIX e siecle, dans le but de demystifier l'intervention de la cesarienne post-mortem de facon a sauver eventuellement le nouveau ne