Valentina Gaidano

Chapter 11: Tissue engineering of peripheral nerves.

Tissue engineering of peripheral nerves has seen an increasing interest over the last years and, similarly to many other fields of regenerative medicine, great expectations have risen within the general public to its potential clinical application in the treatment of damaged nerves. However, in spite of the scientific advancements, applications to the patients is still very limited and it appears that to optimize the strategy for the tissue engineering of the peripheral nerves in the clinical view, researchers have to strive for a new level of innovation which will bring together (in a multitranslational approach) the main pillars of tissue engineering: namely (1) microsurgery, (2) cell and tissue transplantation, (3) material science, and (4) gene transfer. This review paper provides an overview of these four key approaches to peripheral nerve tissue engineering. While some of these issues will also be specifically addressed in other papers in this special issue on peripheral nerve regeneration of the International Review of Neurobiology, in this paper we will focus on an example of successful translational research in tissue engineering, namely nerve reconstruction by muscle-vein-combined nerve scaffolds.

Aberrant activation of ROS1 represents a new molecular defect in chronic myelomonocytic leukemia

Chronic myelomonocytic leukemia (CMML) is a clonal disorder sharing features of myelodysplastic syndromes and chronic myeloproliferative neoplasms. Although rare chromosomal aberrations and point mutations are reported in CMML, the molecular defects underlying CMML are largely unknown. ROS1 encodes a tyrosine kinase that is abnormally expressed and translocated in brain and lung cancers. In this study we show that ROS1 is abnormally activated in the CD34+ compartment of approximately 70% of CMML patients resulting in the activation of the Erk/Akt pathways through the Grb2/SOS complex thus revealing a central oncogenic role for ROS1 in CMML which might represent a molecular target.

Clinical significance of TFR2 and EPOR expression in bone marrow cells in myelodysplastic syndromes

Myelodysplastic syndromes (MDS) are heterogeneous haematopoietic disorders characterized by bone marrow failure, cytopenias and a tendency to transform into acute myeloid leukaemia (AML) (Tefferi & Vardiman, 2009; Vardiman et al, 2009). Scoring systems are used to predict the prognosis of MDS (Greenberg et al, 2012). Percentage of blasts (>10%) and unfavourable cytogenetic abnormalities are the strongest predictors for poor outcome and are associated with high risk or disease progression to AML. Chronic anaemia is the main clinical problem in lowerrisk MDS but responds in 30–50% of cases to erythropoiesisstimulating agents (ESAs). Some prognostic factors of response to ESAs have been identified, with better response rates in patients with no or limited red blood cell transfusion requirement, low baseline serum erythropoietin (EPO) level and no-aberrant myeloid blasts by flow cytometry (Park et al, 2008). Nevertheless, the clinical care of MDS patients is still challenging due to lack of well-established markers that effectively monitor MDS natural history. Therefore, predicting, at diagnosis, those patients with risk of treatment failure is pivotal to personalize treatments in order to improve quality of life and prolong survival. Transferrin receptor 2 (TFR2), homologous to TFR1 (also termed TFRC), is a protein that is mutated in haemochromatosis-type-3 and contributes to the regulation of hepcidin in the liver (Ramos et al, 2011). It is also expressed in erythroid cells and in malignant myeloid disorders (Kawabata et al, 2001). TFR2 associates with erythropoietin receptor (EPOR) (Forejtnikova et al, 2010) and is required for efficient erythropoiesis (Nai et al, 2015). The TFR2 gene gives rise to two isoforms: the full-length TFR2a and the shorter TFR2b (Kawabata et al, 1999). We retrospectively investigated whether TFR2 isoforms and EPOR are differentially expressed in MDS patients and whether the expression is associated with patients’ clinical outcomes. RNA was obtained from the total bone marrow (BM) cells of individuals with non-malignant hematological disorders and in 46 treatment-naive patients at the diagnosis of MDS. After informed consent and ethical approval, TFR2a, TFR2b and EPOR expression was quantified by real-time polymerase chain reaction. Statistical analyses were performed using GraphPad-Prism software (GraphPad Software Inc., La Jolla, CA, USA). Comparison between groups was performed by Mann–Whitney U-test (nonparametric analysis). P values < 0 05 indicated a significant difference. In MDS patients, TFR2a and TFR2b showed higher variability in expression (TFR2a 6 92 5 45; TFR2b 3 16 1 61) than in non-malignant BM cells (TFR2a 8 23 2 97; TFR2b 3 68 0 47). We evaluated TFR2a and TFR2b expression in the different World Health Organization subgroups (Fig 1A–B). TFR2a and TFR2b expression was significantly lower in refractory anaemia with excess, blasts type 2 (RAEB2) (TFR2a 4 44 2 11; TFR2b 2 15 0 59; P < 0 05 and P < 0 01, respectively). A similar expression level was also seen in refractory anaemia with ringed siderobalsts. TFR2a and TFR2b expression was not correlated with total white blood cell, neutrophil and platelet counts, age at diagnosis and no significant differences were observed between sexes (data not shown). We next compared TFR2 expression with that of EPOR. Similarly to TFR2, EPOR expression varied more widely in MDS patients (18 45 18 34) than in non-malignant individuals (17 31 3 97) and was statistically lower in RAEB2 (8 18 2 62, P < 0 005) (Fig 1C). In addition, we found that TFR2a and TFR2b expression was positively correlated with that of EPOR (Fig 1D–E). To assess the clinical implication of this, we analysed the erythroid response in the patients that underwent EPO treatment (Table I). Patients with an increase in haemoglobin of ≥15 g/l after 12 weeks of treatment had TFR2 and/or EPOR levels comparable to normal controls (Fig 1F–G). In contrast, all non-responders had either very high (n = 2) or low (n = 10) TFR2 and EPOR levels. Statistical analysis performed on the 10 TFR2/EPOR low expressing patients showed that the TFR2 and EPOR products were significantly lower compared to EPO responding or non-malignant individuals (Table I). We then tested the effects of TFR2a, TFR2b and EPOR expression on survival in RAEB1-2 and refractory cytopenia with multilineage dysplasia (Fig 1H-J). In the first year of follow-up, patients with very low/low TFR2a or TFR2b expression levels had a significantly worse overall survival than those with normal/high TFR2 expression. Patients in the very low/low EPOR expression group also showed a tendency for poorer survival. Ever since it was demonstrated that TFR2 is a component of the EPOR complex (Forejtnikova et al, 2010), there has been interest in understanding its extra-hepatic function. TFR2 erythroid function has been recently described in normal erythropoiesis in mouse models lacking systemic or Correspondence

Detection of BCR-ABL T315I mutation by peptide nucleic acid directed PCR clamping and by peptide nucleic acid FISH

BackgroundMutations of the BCR-ABL1 fusion gene represent a well established cause of resistance to tyrosine kinase inhibitors. Among the different mutations identified T315I is of particular concern since it is not effectively targeted by the majority of Tyrosine Kinase Inhibitors so far available. We developed a novel assay based on peptide nucleic acid (PNA) technology coupled to immunofluorescence microscopy (PNA-FISH) for the specific detection at a single cell level of BCR-ABLT315I mutation thus improving both, diagnostic resolution and the study of clonal prevalence. Furthermore we developed an additional method based on PNA directed PCR-clamping for the fast and easy detection of the mutation.ResultsThe PNA directed PCR clamping allows to detect an amount of mutated template as low as 0.5 %. This method is highly sensitive, specific and cheap and could be applied even in laboratory not equipped for more sophisticated analysis. Furthermore, the PNA FISH method allows to identify a small amount of progenitor cells still present after therapy with specific inhibitors.ConclusionsWe present here two different methods based on PNA for the detection of T315I useful for different purposes. PNA-FISH can be used to study clonal evolution. In addition, this method could help in the study of compound mutations being able to identify two different mutations in a single cell. PNA directed PCR clamping although not superior to sequencing can be applied worldwide even in laboratory not equipped to search for mutations.

The Wilms’ tumor (WT1) gene expression correlates with the International Prognostic Scoring System (IPSS) score in patients with myelofibrosis and it is a marker of response to therapy

The Wilms tumor gene WT1 is a useful marker of clonal hematopoiesis and it has been shown to be a good marker of residual disease and it reflects the response to therapy. Although myelofibrosis is characterized by mutations of JAK2 and calreticulin (CALR), these mutations are not useful to monitor response to therapy. In this study we demonstrated that in patients affected by myelofibrosis WT1 correlates with the International Prognostic Scoring System (IPSS) score at diagnosis. Furthermore WT1 is a good marker of response to JAK2 inhibitors especially for patients without blasts and for patients who develop anemia or thrombocytopenia not for progression but as therapy related toxicity. Finally, WT1 transcript reduction can mirror a benefit of therapy on the disease burden. This study demonstrated that WT1 is a good marker for monitoring the response to therapy in patients affected by myelofibrosis.

Design and application of a novel PNA probe for the detection at single cell level of JAK2V617F mutation in Myeloproliferative Neoplasms

BackgroundMutation(s) of the JAK2 gene (V617F) has been described in a significant proportion of Philadelphia negative Myeloproliferative Neoplasms (MPN) patients and its detection is now a cornerstone in the diagnostic algorithm.MethodsWe developed a novel assay based on peptide nucleic acid (PNA) technology coupled to immuno-fluorescence microscopy (PNA-FISH) for the specific detection at a single cell level of JAK2-mutation thus improving both the diagnostic resolution and the study of clonal prevalence.ResultsUsing this assay we found a percentage of mutated CD34+ cells ranging from 40% to 100% in Polycythemia Vera patients, from 15% to 80% in Essential Thrombocythemia and from 25% to 100% in Primary Myelofibrosis. This method allows to distinguish, with a high degree of specificity, at single cell level, between CD34+ progenitor stem cells harbouring the mutated or the wild type form of JAK2 in NPM patients.ConclusionsThis method allows to identify multiple gene abnormalities which will be of paramount relevance to understand the pathophysiology and the evolution of any type of cancer.

Prognostic significance of The Wilms’ Tumor-1 (WT1) rs16754 polymorphism in acute myeloid leukemia

Acute myeloid leukemia is a genetically heterogeneous disease characterized by the accumulation of mutations in hematopoietic progenitor cells. For its heterogeneity, prognostic markers are very useful for therapeutic choice. The most important prognostic markers are age, white blood cell count, chromosomal alterations and gene mutations. Recent works have studied the prognostic significance of WT1 polymorphisms and mutations, highlighting the role of SNP rs16754 as a positive prognostic factor in AML patients. Nevertheless, the data are still unclear. To investigate the role of WT1 rs16754 polymorphism in AML, we designed a new tool for the detection using PNA directed PCR Clamping technology. Our data were able to establish a correlation between SNP rs16754 and the clinical outcome. Our results support the hypothesis that rs16754 polymorphism is an independent positive prognostic molecular marker that could be useful for therapeutic choice. In view of this, we described a novel assay faster, more sensitive and cheaper than DNA sequencing. The assay allows evaluating WT1 rs16754 polymorphism in diagnostic routine to improve prognostic information faster and without over-costing for diagnostic laboratories.

Development of cellular and humoral response against WT1 protein vaccination in mice

To the Editor: Many anti‐cancer vaccination strategies have been tested in mice and humans in the attempt to eradicate leukemia cells 1. The vast majority of clinical trials are based on peptide vaccination which allows the induction of cellular response to specific tumor associated antigens 2. WT1(Wilms tumor‐1) gene is located on chromosome 11p13 and encodes a zinc finger transcription factor that plays an important role in cell growth and differentiation. WT1 was originally described as a tumor suppressor gene although many evidences demonstrated that it plays an oncogenic function in the setting of leukemia. WT1 protein represents an optimal tumor antigen since it is highly expressed in acute leukemias, myelodysplastic syndromes (MDS) and myeloproliferative neoplasms 3. By contrast, it is expressed at very low levels in normal hematopoietic progenitors. Expression of the WT1 protein is restricted to a limited set of tissues, including the gonads, uterus, kidney, and spleen.

Abstract 251: Disabled gene is involved in CML progression and its expression level at diagnosis can predict major molecular response (MMR) to imatinib therapy

Despite the role of Bcr-Abl in the pathogenesis of Chronic Myeloid Leukemia (CML) is well established, the mechanisms leading to CML progression remain unknown. Using our model of Drosophila melanogaster (Dm) transgenic for human Bcr-Abl we identified Dab1 and Dab2, the homologs of Dm Disabled (Dab), as genes involved in CML progression. In Dm the Dab loss of function induced a worsening of the hBcr-Abl eye phenotype and an even stronger phenotype was obtained using Dab RNAi fly strains. By contrast, Dab gain of function rescued Bcr-Abl phenotype. Dab is an adaptor protein acting downstream of many receptor tyrosine kinases (RTK). One of the human homolog of Dab, Dab1 is a large common fragile site gene involved in neural migration, and the other homolog Dab2 encodes an adaptor protein implicated in RTK signalling, endocytosis, cell adhesion and differentiation. The downregulation of both genes is described in many cancers suggesting their possible role in oncogenesis but their involvement in haematological malignancies has never been described. The aim of the study was to investigate the role of Dab1/2 in CML progression. Dab1 and Dab2 mRNA was analyzed by Real Time PCR in 94 samples from 82 CML patients (34 PB and 60 BM) distributed as follows: 55 patients at diagnosis (19 enrolled in TOPS study), 9 chronic phase (CP), 7 accelerated phase (AP) and 11 blast crisis (BC). 21 healthy donors (10 PB and 11 BM) were analyzed as control. In 18 patients, genes expression was analyzed during remission as well. Protein expression was evaluated by Western Blot (WB) and Immunofluorescence (IF). In addition, K562 cells were transfected with Dab plasmids to evaluate the effects on cell proliferation. We found that in CML patients Dab1/2 expression was significantly decreased both in BM and PB (p Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 251.

Abstract 246: Genome-wide screening for dominant modifiers in Drosophila identified new genes involved in BCR-ABL signaling and chronic myeloid leukemia (CML) progression

Despite the role of Bcr-Abl in the pathogenesis of CML is well established, the mechanisms responsible for CML progression remain unknown. The aim of the study was to perform a genome-wide screening to identify new genes and pathways leading to CML progression. We performed the genetic screening using our set-up model of human p210 Bcr-Abl (hBcr-Abl) transgenic Drosophila melanogaster (Dm) in which the tissue specific expression of hBcr-Abl is able to induce a severe eye phenotype or the formation of melanotic tumors, when expressed into the fly lymph gland, the Dm hematopoietic system. The whole fly genome containing 14.000 genes was analyzed using 278 fly stocks carrying well characterized chromosome deletions. The resulting progeny was screened using eye and lymph gland as first and second read-out system respectively. Data obtained from both screens were analyzed using the Gene Ontology software. These results were compared with gene expression signatures of CML from Microarray data. As final point, the identified genes were validated analyzing either BM or PB samples from CML patients and healthy donors. The analysis of eye/lymph gland-phenotypes in the progeny obtained from screen crosses, shows a first group of flies (38%) displaying a more aggressive phenotype since they lack genes encoding for hBcr-Abl negative regulators and a second group (32%) showing a mild phenotype due to the absence of genes involved in the oncogenic signalling. 42% of the 4000 Dm genes mapping in these regions displayed a known human counterpart. Gene Ontology profiles of these genes included oncogenes, tumor suppressors and genes involved in the regulation of transcription, signal transduction, proliferation, differentiation, apoptosis and splicing. Moreover, a computational comparison of our results in fly with gene expression signatures of CML from Microarray data, showed only a partial overlap. Interestingly, the 72% of identified genes was not known to be involved in human leukaemia. However, further confirmation of our findings comes from the validation in human samples in which 1250 genes were found to be associated with CML; among these genes, we found not only an alteration of their expression profiles in CML patients compared to the healthy donors, but also protein alterations such as expression of different splicing forms or misplaced proteins, suggesting that Dm screening is a valid approach to identify not only differentially expressed genes but also specific pathways and genes otherwise altered by hBcr-Abl. In conclusion, the identification of these genes allows identifying of the changes occurring in CML at the genomic level and gives deeper insights into the molecular basis of the disease; moreover this study points to specific gene pathways that might represent new targets for therapy in CML in order to prevent or overcome resistance and progression. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 246.