J S Wainscoat

University of Oxford, Oxford, England, United Kingdom

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Publications (272)2639.65 Total impact

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    ABSTRACT: The diagnosis of patients with myelodysplastic syndromes (MDS) is largely dependent on morphologic examination of bone marrow aspirates. Several criteria that form the basis of the classifications and scoring systems most commonly used in clinical practice are affected by operator-dependent variation. To identify standardized molecular markers that would allow prediction of prognosis, we have used gene expression profiling (GEP) data on CD34+ cells from patients with MDS to determine the relationship between gene expression levels and prognosis. GEP data on CD34+ cells from 125 patients with MDS with a minimum 12-month follow-up since date of bone marrow sample collection were included in this study. Supervised principal components and lasso penalized Cox proportional hazards regression (Coxnet) were used for the analysis. We identified several genes, the expression of which was significantly associated with survival of patients with MDS, including LEF1, CDH1, WT1, and MN1. The Coxnet predictor, based on expression data on 20 genes, outperformed other predictors, including one that additionally used clinical information. Our Coxnet gene signature based on CD34+ cells significantly identified a separation of patients with good or bad prognosis in an independent GEP data set based on unsorted bone marrow mononuclear cells, demonstrating that our signature is robust and may be applicable to bone marrow cells without the need to isolate CD34+ cells. We present a new, valuable GEP-based signature for assessing prognosis in MDS. GEP-based signatures correlating with clinical outcome may significantly contribute to a refined risk classification of MDS.
    Preview · Article · Sep 2013 · Journal of Clinical Oncology
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    ABSTRACT: Interstitial deletion of chromosome 5q is the most common chromosomal abnormality in myelodysplastic syndromes. The catalogue of genes involved in the molecular pathogenesis of myelodysplastic syndromes is rapidly expanding and next-generation sequencing technology allows detection of these mutations at high depth. Here we describe the design, validation and application of a targeted next-generation sequencing approach to simultaneously screen 25 genes mutated in myeloid malignancies. We used this method alongside single nucleotide polymorphism-array technology to characterize the mutational and cytogenetic profile of 43 early or advanced del(5q) myelodysplastic syndrome cases. A total of 29 mutations were detected in our cohort. Overall, 45% of early and 66.7% of advanced cases presented at least one mutation. Genes with the highest mutation frequency among advanced cases were TP53 and ASXL1 (25% of patients each). These showed a lower mutation frequency in 5q- syndrome cases (4.5% and 13.6%, respectively), suggesting a role in disease progression in del(5q) myelodysplastic syndromes. 52% of mutations identified were in genes involved in epigenetic regulation (ASXL1, TET2, DNMT3A and JAK2). Six mutations showed allele frequencies <20%, likely below the detection limit of traditional sequencing methods. Genomic array data showed that advanced del(5q) myelodysplastic syndrome cases displayed a complex background of cytogenetic aberrations, often encompassing genes involved in myeloid disorders. Our study is the first to investigate the molecular pathogenesis of early and advanced del(5q) myelodysplastic syndromes using next-generation sequencing technology on a large panel of genes frequently mutated in myeloid malignancies, further illuminating the molecular landscape of del(5q) myelodysplastic syndromes.
    Full-text · Article · Jul 2013 · Haematologica
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    ABSTRACT: Leukemia is one of the leading journals in hematology and oncology. It is published monthly and covers all aspects of the research and treatment of leukemia and allied diseases. Studies of normal hemopoiesis are covered because of their comparative relevance.
    No preview · Article · Jan 2013 · Leukemia: official journal of the Leukemia Society of America, Leukemia Research Fund, U.K
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    ABSTRACT: The ASXL1 gene encodes a chromatin-binding protein involved in epigenetic regulation in haematopoietic cells. Loss-of-function ASXL1 mutations occur in patients with a range of myeloid malignancies and are associated with adverse outcome. We have used lentiviral-based shRNA technology to investigate the effects of ASXL1 silencing on cell proliferation, apoptosis, myeloid differentiation and global gene expression in human CD34(+) cells differentiated along the myeloid lineage in vitro. ASXL1-deficient cells showed a significant decrease in the generation of CD11b(+) and CD15(+) cells, implicating impaired granulomonocytic differentiation. Furthermore, colony-forming assays showed a significant increase in the number of multipotent mixed lineage colony-forming unit (CFU-GEMM) colonies and a significant decrease in the numbers of granulocyte-macrophage CFU (CFU-GM) and granulocyte CFU (CFU-G) colonies in ASXL1-deficient cells. Our data suggests that ASXL1 knockdown perturbs human granulomonocytic differentiation. Gene expression profiling identified many deregulated genes in the ASXL1-deficient cells differentiated along the granulomonocytic lineage, and pathway analysis showed that the most significantly deregulated pathway was the LXR/RXR activation pathway. ASXL1 may play a key role in recruiting the polycomb repressor complex 2 (PRC2) to specific loci, and we found over-representation of PRC2 targets among the deregulated genes in ASXL1-deficient cells. These findings shed light on the functional role of ASXL1 in human myeloid differentiation.
    Full-text · Article · Jan 2013 · British Journal of Haematology
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    ABSTRACT: 171 The 5q-syndrome is the most distinct of all myelodysplastic syndromes with a clear genotype/phenotype relationship. Haploinsufficiency of the ribosomal protein gene RPS14 underlies the erythroid defect found in the 5q-syndrome. The 5q-syndrome is a disorder of aberrant ribosome biogenesis and we have recently demonstrated that erythrocytes obtained from patients with the 5q-syndrome show impaired translation. This defect in translation may represent a potential therapeutic target in the 5q-syndrome and other ribosomopathies, and there are some indications that the use of the translation enhancer L-leucine may have some efficacy. L-leucine is a branched chain amino acid that has been shown to improve haemoglobin levels and transfusion independence in patients with the ribosomopathy Diamond Blackfan Anemia (DBA). Moreover, the treatment of zebrafish models of DBA and the 5q-syndrome with L-leucine has recently been shown by others to result in partial reversal of the anemia. To model the RPS14 haploinsufficiency observed in the 5q-syndrome, we used lentivirally delivered shRNA sequences to reduce the expression of RPS14 in human bone marrow CD34 + cells from healthy controls. We have recently shown that treatment of cultured human erythroid cells derived from CD34 + cells of healthy controls with RPS14 knockdown and cultured erythroid cells derived from the CD34 + cells of patients with the 5q-syndrome with L-leucine results in an increase in cell proliferation, erythroid differentiation and mRNA translation. There is evidence to suggest that L-leucine activates the mTOR (mammalian target of rapamycin) signaling pathway that controls many cellular processes including cell growth and mRNA translation. In order to investigate the mechanism of action of L-leucine, we have studied the phosphorylation levels of S6K1 and 4EBP1, the key downstream targets of mTORC1 (mTOR Complex 1), by sandwich ELISA, Human Phospho-kinase Antibody Array and flow cytometry in RPS14-deficient human erythroblasts. We have shown a significant increase in the levels of phospho-S6K1 and phospho-4EBP1 following L-leucine treatment of cultured erythroid cells derived from CD34 + cells of healthy controls with RPS14 knockdown. The effects of L-leucine on phospho-S6K1 and phospho-4EBP1 were abrogated by rapamycin (an mTOR inhibitor), suggesting that L-leucine has a specific action on the mTOR signaling pathway. Consistent to the results observed in the RPS14 knockdown model, treatment with L-leucine also increased the level of phospho-S6K1 in cultured erythroid cells derived from the bone marrow cells of 5q-MDS patients. These data suggest that L-leucine activates the mTOR pathway in RPS14-deficient human erythroblasts. Our studies support the evaluation of L-leucine as a potential therapeutic agent in the treatment of the 5q-syndrome, and provide evidence on its mode of action through activation of the mTOR signaling pathway.
    Full-text · Conference Paper · Dec 2012
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    ABSTRACT: Patients with the 5q- syndrome and Diamond-Blackfan anemia (DBA) suffer from a severe macrocytic anemia. The 5q- syndrome and DBA are disorders of aberrant ribosome biogenesis (ribosomopathies) and haploinsufficiency of the ribosomal protein genes RPS14 and RPS19, respectively, underlies the anemia found in these disorders. Erythroblasts obtained from patients with the 5q- syndrome and DBA show impaired mRNA translation and this defect in translation may represent a potential therapeutic target in these ribosomopathies. There are some indications that the amino acid l-leucine, a translation enhancer, may have some efficacy in this group of disorders. Recent studies have shown that l-leucine treatment of zebrafish and murine models of the 5q- syndrome and DBA results in a marked improvement in the anemia. l-leucine treatment of RPS14-deficient and RPS19-deficient erythroblasts and erythroblasts from patients with the 5q- syndrome has been shown to result in an increase in cell proliferation, erythroid differentiation and mRNA translation in culture. l-leucine has been shown to improve hemoglobin levels and transfusion independence in a patient with DBA. l-leucine activates the mTOR (mammalian target of rapamycin) signaling pathway that controls cell growth and mRNA translation. There is evidence to suggest that the promotion of translation via the mTOR pathway by l-leucine is the mechanism that underlies the enhanced erythroid progenitor cell growth and differentiation observed in animal and cellular models of the 5q- syndrome and DBA treated with this amino acid. These data support the rationale for clinical trials of l-leucine as a therapeutic agent for the 5q- syndrome and DBA.
    No preview · Article · Sep 2012
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    ABSTRACT: Acute myeloid leukemia patients with normal cytogenetics (CN-AML) account for almost half of AML cases. We aimed to study the frequency and relationship of a wide range of genes previously reported as mutated in AML (ASXL1, NPM1, FLT3, TET2, IDH1/2, RUNX1, DNMT3A, NRAS, JAK2, WT1, CBL, SF3B1, TP53, KRAS and MPL) in a series of 84 CN-AML cases. The most frequently mutated genes in primary cases were NPM1 (60.8%) and FLT3 (50.0%), and in secondary cases ASXL1 (48.5%) and TET2 (30.3%). We showed that 85% of CN-AML patients have mutations in at least one of ASXL1, NPM1, FLT3, TET2, IDH1/2 and/or RUNX1. Serial samples from 19 MDS/CMML cases that progressed to AML were analyzed for ASXL1/TET2/IDH1/2 mutations; seventeen cases presented mutations of at least one of these genes. However, there was no consistent pattern in mutation acquisition during disease progression. This report concerns the analysis of the largest number of gene mutations in CN-AML studied to date, and provides insight into the mutational profile of CN-AML.
    Full-text · Article · Aug 2012 · PLoS ONE
  • Jacqueline Boultwood · Andrea Pellagatti · James S Wainscoat
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    ABSTRACT: In recent years we have gained great insight into the molecular pathogenesis of the 5q- syndrome, the most distinct of all the myelodysplastic syndromes. It is now recognized that p53 activation, caused by haploinsufficiency for the ribosomal gene RPS14 (mapping to the commonly deleted region), is the probable cause of the erythroid defect in the 5q- syndrome. A mouse model of the human 5q- syndrome has been generated by large-scale deletion of the Cd74-Nid67 interval (containing Rps14) and the crossing of these '5q- mice' with p53-deficient mice ameliorated the erythroid progenitor defect. Recent evidence suggests that haploinsufficiency of the microRNA genes miR-145 and miR-146a may contribute to the thrombocytosis seen in the 5q- syndrome. Emerging data shows that p53 mutation may play a role in disease progression.
    No preview · Article · May 2012 · Current pharmaceutical design
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    ABSTRACT: Leukemia is one of the leading journals in hematology and oncology. It is published monthly and covers all aspects of the research and treatment of leukemia and allied diseases. Studies of normal hemopoiesis are covered because of their comparative relevance.
    Full-text · Article · Mar 2012 · Leukemia: official journal of the Leukemia Society of America, Leukemia Research Fund, U.K
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    ABSTRACT: Chronic myelomonocytic leukemia (CMML) has recently been associated with a high incidence of diverse mutations in genes such as TET2 or EZH2 that are implicated in epigenetic mechanisms. We have performed genome-wide DNA methylation arrays and mutational analysis of TET2, IDH1, IDH2, EZH2 and JAK2 in a group of 24 patients with CMML. 249 genes were differentially methylated between CMML patients and controls. Using Ingenuity pathway analysis, we identified enrichment in a gene network centered around PLC, JNK and ERK suggesting that these pathways, whose deregulation has been recently described in CMML, are affected by epigenetic mechanisms. Mutations of TET2, JAK2 and EZH2 were found in 15 patients (65%), 4 patients (17%) and 1 patient (4%) respectively while no mutations in the IDH1 and IDH2 genes were identified. Interestingly, patients with wild type TET2 clustered separately from patients with TET2 mutations, showed a higher degree of hypermethylation and were associated with higher risk karyotypes. Our results demonstrate the presence of aberrant DNA methylation in CMML and identifies TET2 mutant CMML as a biologically distinct disease subtype with a different epigenetic profile.
    Full-text · Article · Feb 2012 · PLoS ONE
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    ABSTRACT: In a previous study, we identified somatic mutations of SF3B1, a gene encoding a core component of RNA splicing machinery, in patients with myelodysplastic syndrome (MDS). Here, we define the clinical significance of these mutations in MDS and myelodysplastic/myeloproliferative neoplasms (MDS/MPN). The coding exons of SF3B1 were screened using massively parallel pyrosequencing in patients with MDS, MDS/MPN, or acute myeloid leukemia (AML) evolving from MDS. Somatic mutations of SF3B1 were found in 150 of 533 (28.1%) patients with MDS, 16 of 83 (19.3%) with MDS/MPN, and 2 of 38 (5.3%) with AML. There was a significant association of SF3B1 mutations with the presence of ring sideroblasts (P < .001) and of mutant allele burden with their proportion (P = .002). The mutant gene had a positive predictive value for ring sideroblasts of 97.7% (95% confidence interval, 93.5%-99.5%). In multivariate analysis including established risk factors, SF3B1 mutations were found to be independently associated with better overall survival (hazard ratio = 0.15, P = .025) and lower risk of evolution into AML (hazard ratio = 0.33, P = .049). The close association between SF3B1 mutations and disease phenotype with ring sideroblasts across MDS and MDS/MPN is consistent with a causal relationship. Furthermore, SF3B1 mutations are independent predictors of favorable clinical outcome, and their incorporation into stratification systems might improve risk assessment in MDS.
    Preview · Article · Dec 2011 · Blood
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    ABSTRACT: Myelodysplastic syndromes are a diverse and common group of chronic hematologic cancers. The identification of new genetic lesions could facilitate new diagnostic and therapeutic strategies. We used massively parallel sequencing technology to identify somatically acquired point mutations across all protein-coding exons in the genome in 9 patients with low-grade myelodysplasia. Targeted resequencing of the gene encoding RNA splicing factor 3B, subunit 1 (SF3B1), was also performed in a cohort of 2087 patients with myeloid or other cancers. We identified 64 point mutations in the 9 patients. Recurrent somatically acquired mutations were identified in SF3B1. Follow-up revealed SF3B1 mutations in 72 of 354 patients (20%) with myelodysplastic syndromes, with particularly high frequency among patients whose disease was characterized by ring sideroblasts (53 of 82 [65%]). The gene was also mutated in 1 to 5% of patients with a variety of other tumor types. The observed mutations were less deleterious than was expected on the basis of chance, suggesting that the mutated protein retains structural integrity with altered function. SF3B1 mutations were associated with down-regulation of key gene networks, including core mitochondrial pathways. Clinically, patients with SF3B1 mutations had fewer cytopenias and longer event-free survival than patients without SF3B1 mutations. Mutations in SF3B1 implicate abnormalities of messenger RNA splicing in the pathogenesis of myelodysplastic syndromes. (Funded by the Wellcome Trust and others.).
    Full-text · Article · Oct 2011 · New England Journal of Medicine
  • Jacqueline Boultwood · Andrea Pellagatti · James S Wainscoat

    No preview · Article · Sep 2011 · Advances in enzyme regulation
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    Dataset: Table S1
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    ABSTRACT: Using a one-sided hypothesis test, with a null hypothesis that p1 = p2, and an alternative hypothesis that p1<p2, at 99% confidence, the critical test statistic is 2.33. Since the test statistics above are all greater than 2.33, then we can reject the null hypothesis in all cases. Note: it is often recommended that this test only be applied when both the number of successes and the number of failures is at least 5 in both populations. Although this is strictly not satisfied in population 2 above, it is satisfied for the cell population as a whole. Also, if the number of failures in population 2 (i.e. the number of Ki-67 positive cells with 3 signals) was 5 for all patients (a result which is clearly not as significant as the result above), then the result would still be statistically significant at 99% confidence for all patients except patient 5. For patient 5 it would be statistically significant at 98% confidence. (DOC)
    Preview · Dataset · Jun 2011
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    ABSTRACT: Leukaemia is often associated with genetic alterations such as translocations, amplifications and deletions, and recurrent chromosome abnormalities are used as markers of diagnostic and prognostic relevance. However, a proportion of acute myeloid leukaemia (AML) cases have an apparently normal karyotype despite comprehensive cytogenetic analysis. Based on conventional cytogenetic analysis of banded chromosomes, we selected a series of 23 paediatric patients with acute myeloid leukaemia and performed whole genome array comparative genome hybridization (aCGH) using DNA samples derived from the same patients. Imbalances involving large chromosomal regions or entire chromosomes were detected by aCGH in seven of the patients studied. Results were validated by fluorescence in situ hybridization (FISH) to both interphase nuclei and metaphase chromosomes using appropriate bacterial artificial chromosome (BAC) probes. The majority of these copy number alterations (CNAs) were confirmed by FISH and found to localize to the interphase rather than metaphase nuclei. Furthermore, the proliferative states of the cells analyzed by FISH were tested by immunofluorescence using an antibody against the proliferation marker pKi67. Interestingly, these experiments showed that, in the vast majority of cases, the changes appeared to be confined to interphase nuclei in a non-proliferative status.
    Full-text · Article · Jun 2011 · PLoS ONE
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    Dataset: Figure S1
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    ABSTRACT: Examples of FISH using a centromeric probe specific for chromosome 7 (7cen) on patient no. 1. Two red signals are clearly visible in the metaphase, whereas three fluorescent signals are present in the nucleus in the same sample. (TIF)
    Preview · Dataset · Jun 2011
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    ABSTRACT: MicroRNAs are small RNA species that regulate gene expression post-transcriptionally and are aberrantly expressed in many cancers including hematological malignancies. However, the role of microRNAs in the pathogenesis of multiple myeloma (MM) is only poorly understood. We therefore used microarray analysis to elucidate the complete miRNome (miRBase version 13.0) of purified tumor (CD138+) cells from 33 patients with MM, 5 patients with monoclonal gammopathy of undetermined significance (MGUS) and 9 controls. Unsupervised cluster analysis revealed that MM and MGUS samples have a distinct microRNA expression profile from control CD138+ cells. The majority of microRNAs aberrantly expressed in MM (109/129) were up-regulated. A comparison of these microRNAs with those aberrantly expressed in other B-cell and T-cell malignancies revealed a surprising degree of similarity (~40%) suggesting the existence of a common lymphoma microRNA signature. We identified 39 microRNAs associated with the pre-malignant condition MGUS. Twenty-three (59%) of these were also aberrantly expressed in MM suggesting common microRNA expression events in MM progression. MM is characterized by multiple chromosomal abnormalities of varying prognostic significance. We identified specific microRNA signatures associated with the most common IgH translocations (t(4;14) and t(11;14)) and del(13q). Expression levels of these microRNAs were distinct between the genetic subtypes (by cluster analysis) and correctly predicted these abnormalities in > 85% of cases using the support vector machine algorithm. Additionally, we identified microRNAs associated with light chain only myeloma, as well as IgG and IgA-type MM. Finally, we identified 32 microRNAs associated with event-free survival (EFS) in MM, ten of which were significant by univariate (logrank) survival analysis. In summary, this work has identified aberrantly expressed microRNAs associated with the diagnosis, pathogenesis and prognosis of MM, data which will prove an invaluable resource for understanding the role of microRNAs in this devastating disease.
    Full-text · Article · May 2011 · Biology Direct
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    ABSTRACT: Additional tables (Tables S1-S7) and figure (Figures S1-S4). Table S1. Clinical details of MM and MGUS patients. Table S2. Table of microRNAs aberrantly expressed in DLBCL, SzS and MM, highlighting those that are common. Table S3. MicroRNAs differentially expressed (P < 0.05) between MGUS (n = 5) and controls (n = 9). Table S4. MicroRNAs differentially expressed (P < 0.05) between MM (n = 32) and MGUS (n = 5). Table S5. MicroRNAs differentially expressed (P < 0.05) between IgA (n = 8) and IgG (n = 13) isotype MM cases. Table S6. MicroRNAs differentially expressed (P < 0.05) between LC-only myeloma (n = 8) and non-LC-only myeloma MM cases (n = 21). Table S7. MicroRNAs associated with event-free survival (EFS) in MM cases. Figure S1. Heat map depicting cluster analysis of MM and control samples on the basis of expression values of 129 MM-associated microRNAs. Figure S2. Heat map depicting cluster analysis of MGUS and control samples on the basis of expression values of 39 MGUS-associated microRNAs. Figure S3. Venn-diagram depicting relationship between microRNAs differentially expressed in MGUS, MM and controls. Figure S4. Heat map depicting cluster analysis of IgG and IgA isotype MM cases on the basis of expression values of 21 isotype-associated microRNAs (Table S5).
    Preview · Dataset · May 2011

  • No preview · Article · May 2011 · Leukemia Research
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    Full-text · Article · Apr 2011 · British Journal of Haematology

Publication Stats

15k Citations
2,639.65 Total Impact Points

Institutions

  • 2003-2013
    • University of Oxford
      • • Nuffield Division of Clinical Laboratory Sciences
      • • Molecular Haematology Unit
      Oxford, England, United Kingdom
  • 1981-2013
    • Oxford University Hospitals NHS Trust
      • • Department of Haematology
      • • Nuffield Department of Clinical Laboratory Sciences
      • • Nuffield Department of Medicine
      Oxford, England, United Kingdom
    • Hospital de Base
      San Paulo, São Paulo, Brazil
  • 2007
    • University of Pavia
      Ticinum, Lombardy, Italy
    • Dana-Farber Cancer Institute
      • Department of Medical Oncology
      Boston, Massachusetts, United States
  • 2001
    • Prince of Wales Hospital, Hong Kong
      Chiu-lung, Kowloon City, Hong Kong
  • 1999
    • The Chinese University of Hong Kong
      • Department of Chemical Pathology
      Hong Kong, Hong Kong
  • 1988
    • University of Leicester
      Leiscester, England, United Kingdom
  • 1986
    • The University of the West Indies at Mona
      Kingston, Kingston, Jamaica
    • University College Hospital Ibadan
      Ibadan, Oyo, Nigeria
    • Boston Children's Hospital
      Boston, Massachusetts, United States
  • 1985
    • Freie Universität Berlin
      • Institute of Veterinary Pathology
      Berlín, Berlin, Germany