ALBERT

Description: Abstract 1999 Poster Board I-1021 Background: Recent evidence suggests the involvement of lymphocytes in the severity of iron overload disorders, with increased severity of iron overload in Hereditary Hemochromatosis patients and in animal models with lymphocyte number deficiencies. However, no mechanism(s) has been suggested to explain these observations. The aim of this study was to analyze hepcidin expression in lymphocytes. Methods: Expression of hepcidin was analyzed by Real-time PCR in human and mouse Peripheral Blood Lymphocytes (PBLs) and in selected resting lymphocyte populations, in response to holotransferrin and ferric citrate. The effect of hepcidin in the expression of the iron exporter Ferroportin was analyzed by FACS and confocal immunofluorescence. Cellular iron traffic was analyzed by measurement of 55Fe and 125I-TF, cell proliferation assessed by BrdU incorporation and silencing of gene expression in lymphocytes performed with siRNAs. Results: Hepcidin is expressed in PBLs and is up-regulated in response to holotransferrin and ferric citrate. The response to holotransferrin was observed in CD8+ and not in CD4+ lymphocytes, a result confirmed by the failure of lymphocytes from β2-microglobulin-KO mice to respond to holotransferrin, in comparison with Bl6/J controls. Hepcidin up-regulation induced by holotransferrin decreases ferroportin expression, inducing its co-localization with the proteasome marker LMP2. Tumor Necrosis Factor-á (TNF-á) expression in PBLs increases with holotransferrin treatments. siRNA-mediated silencing of TNF-á in PBLs abrogates hepcidin up-regulation by holotransferrin and incubation of PBLs with recombinant TNF-á increases hepcidin expression, suggesting the involvement of this cytokine in the basal and holotransferrin-induced hepcidin expression in these cells. The role of hepcidin in a situation of high iron demand - lymphocyte activation and proliferation - was assessed. Hepcidin expression increases with T-lymphocyte activation and siRNA-mediated silencing of hepcidin in activated T lymphocytes causes a decrease in intracellular iron levels, by increasing ferroportin-mediated iron export. The low intracellular iron levels were associated with impaired T lymphocyte proliferation. Discussion: The ability of PBLs to increase hepcidin expression in response to ferric citrate distinguishes lymphocytes from hepatocytes and places peripheral blood lymphocyte numbers as a first line of response to increases in transferrin saturation and presence of NTBI, characteristic of iron overload disorders. The findings that hepcidin modulates ferroportin expression, intracellular iron levels and cell proliferation in lymphocytes demonstrate the importance of this protein for lymphocyte iron homeostasis. The control of the intracellular iron levels of lymphocytes confers to hepcidin a pivotal role in the postulated ability of circulating lymphocytes to function as “biological iron chelators”. Conclusion: With the demonstration, for the first time, of hepcidin synthesis by peripheral blood lymphocytes, of its regulation by elemental iron and of its involvement in T cell proliferation, the present results put forward a molecular mechanism for the described modifier role of lymphocytes in protection from iron toxicity. Disclosures: No relevant conflicts of interest to declare.

Description: Human chromosome translocations at 11q23, disrupting the MLL1 gene, result in poor prognostic mixed lineage leukaemias. Current chemotherapy treatment protocols produce an unsatisfactory outcome. Indeed, the average five-year event free survival rate is 44% in paediatric cases, and adult cases have been estimated as low as 15% for two-year survival rates, indicating there is an unmet critical need for more effective therapies. In recent years, there has been great interest in targeting the epigenetic factors involved in MLL-rearranged (MLL-r) leukaemic transformation and maintenance; however, epigenetic plasticity, the potential role of the remaining MLL1 allele and the elusive leukaemic stem cells present in acute myeloid leukaemia (AML), provide many routes to chemoresistance. There is currently great interest in targeting the cell cycle and key intracellular signalling pathways (e.g. Wnt signalling), independent of specific aberrant lesions in AML (e.g. MLL-fusion proteins, DNMT3a mutants), to combat highly quiescent leukaemic stem cells, which are the most difficult to eradicate. In addition, protection of the resident normal haematopoietic stem cells (HSCs), during aggressive induction chemotherapy protocols, provides another route to reduce the competitive advantage of AML cells in vivo. We previously identified two new genes, involved in the regulation of MLL1, Wnt signalling and the cell cycle: the CDK subunits CKS1 and CKS2 (Grey et al. 2017). Here, we investigated the roles of CKS1 and CKS2 during normal and malignant haematopoiesis in vivo, revealing differences in key signalling pathways involved in haematopoiesis and leukaemogenesis, implicating the CKS1/CKS2 axis as a valid therapeutic target. We demonstrate that primary AML patient samples, engrafted in immune deficient mice, are sensitive to inhibition of CKS1-dependent protein degradation, with reduced tumour burden after treatment and significant improvement in survival times. In addition, patient samples showed CKS1-sensitivity irrespective of inherent resistance to Cytarabine. Current chemotherapy protocols, using Cytarabine and Doxorubicin, can be significantly deleterious to resident normal HSCs in vivo. Transient inhibition of CKS1-dependent protein degradation, in vivo, provides a protective function to human CD34+ HSPCs when treated with Cytarabine/Doxorubicin (5+3 dosing protocol), resulting in reduced apoptosis and increased stem cell potential post-therapy. Importantly, combination treatment of CKS1 inhibition with Cytarabine/Doxorubicin significantly reduces AML tumour burden and improves overall survival, by selectively killing AML cells and preserving normal resident HSCs. Altogether, these results open a promising alternative approach for modulating protein phosphorylation and degradation to selectively target leukaemic cells, with the great advantage to protect normal resident HSCs from cytotoxic effects of induction chemotherapy. Disclosures No relevant conflicts of interest to declare.

Description: Introduction Protein kinases play a key role in how cells respond and adapt to intra and extracellular stimuli. By the addition of phosphate groups to serine, threonine or tyrosine residues, these enzymes modify the activity and properties of the targeted proteins which in turn modulate biological processes like proliferation, differentiation and cell death. Kinase signalling pathways are deregulated in most cancer types including haematological malignancies. Indeed, the kinases FLt-3, c-Kit and JAK2 as well as the up-stream kinase signalling regulators KRAS and NRAS are among the most frequently mutated genes in acute myeloid leukaemia (AML). Consequently, protein kinases have attracted the attention of the pharmaceutical and biotechnology companies and inhibitors have been found for one fifth of human kinases. In the case of AML, midostaurin, a multi-kinase inhibitor that targets, among others, the tyrosine kinase Flt-3, has granted a breakthrough therapy designation by the FDA and several other kinase inhibitors are in clinical trials or under preclinical investigation. Molecular profiling of patient samples will play a pivotal role for the development and implementation of personalized therapies including those based on kinase inhibitors. We used a molecular profile generated by a phosphoproteomics approach to rationalize why some primary AML cells respond to treatment with different kinase inhibitors while others are resistant to the same treatments. Methods Label free phosphoproteomics based on trypsin digestion and TiO2 phosphoenrichment was used to quantify 〉 5,000 phosphorylation sites in mononuclear cells extracted from the peripheral blood of 36 AML patients. KSEA technology was applied to infer kinase activity from the phosphoproteomics data and DAVID software was used to determine gene ontology enrichments based on the genes that code for the proteins where the phosphorylation sites were detected. Guava EasyCyte Flow Cytometry was used to determine cell viability after the treatment of the same patient samples with different kinase inhibitors. Mass cytometry was used to measure the expression at the plasma membrane of 17 surface markers in 30 of the previously analysed AML primary samples. Results The FAB classification subdivide AML cases depending on cytomorphological features. We compared the phosphoproteomes of M1 and M4 classes that are associated with early and late states of differentiation. Based on the 150 phosphopeptides more significantly regulated between FAB-M1 and FAB-M4 groups, hierarchical clustering analysis was used to stratify AML patient samples into two subsets named M1-Like and M4-Like. Phosphoproteome reanalysis showed that the M4-Like set upregulated 1255 phosphopeptides and downregulated 446 when compared with the M1-Like set. The upregulated group comprised regulatory phosphorylation sites in several kinases including PAK1 and PCK delta. Kinase activity analysis using KSEA (Kinase Substrate Enrichment Analysis) also showed an increased activity of PAK, PKCδ and other kinases like P38 alpha in the M4-Like group. Interestingly, the PAK inhibitor PF03758309 reduced more efficiently the viability in M4-Like group than in the M1-Like group (average reduction after a 72h treatment with 1µM of 55.2% for M4-Like compared to 33.8% for M1-Like, p-value = 0.0078). This difference was not observed for other inhibitors such as those targeting CK2 or p38. CyTOF analysis showed that the M4-Like group upregulated the surface expression of several differentiation markers. Discussion Predicting the effectiveness of a drug for a particular patient is a major goal of personalized medicine. In the case of kinase inhibitors, responses may be influenced by several factors including the activity of the targeted kinase as well as the activity of other kinases that act in parallel pro-survival pathways. In this work, we have found that differentiation leads to a particular activation pattern of the signalling networks, a phenomenon that determines the response to signalling inhibitors. Conclusion We found phosphoproteomics signatures in primary AML that are associated with distinct haematopoietic differentiation stages. These signatures are in turn associated with how AML cells respond to kinase inhibitors. Disclosures Fitzgibbon: Epizyme: Research Funding; Gilead: Honoraria; Janssen: Honoraria; Celgene: Honoraria.

Description: Mantle cell lymphoma (MCL) is molecularly characterized by bcl-1 rearrangement and cyclin D1 gene overexpression. Some aggressive variants of MCL have been described with blastic or large cell morphology, higher proliferative activity, and shorter survival. The cyclin-dependent kinase inhibitors (CDKIs) p21Waf1 and p16INK4a have been suggested as candidates for tumor-suppressor genes. To determine the role of p21Waf1 and p16INK4a gene alterations in MCLs, we examined the expression, deletions, and mutations of these genes in a series of 24 MCLs, 18 typical, and 6 aggressive variants. Loss of expression and/or deletions of p21Waf1 and p16INK4a genes were detected in 4 (67%) aggressive MCLs but in none of the typical variants. Two aggressive MCLs showed a loss of p16INK4a expression. These cases showed homozygous deletions of p16INK4a gene by Southern blot analysis. An additional aggressive MCL in which expression could not be examined showed a hemizygous 9p12 deletion. Loss of p21Waf1 expression at both protein and mRNA levels was detected in an additional aggressive MCL. No p21Waf1 gene deletions or mutations were found in this case. The p21Waf1 expression in MCLs was independent of p53 mutations. The two cases with p53 mutations showed p21Waf1 and p16INK4a expression whereas the 4 aggressive MCLs with p16INK4a and p21Waf1 gene alterations had a wild-type p53. p21Waf1 and p16INK4a were expressed at mRNA and protein levels in all typical MCLs examined. No gene deletions or point mutations were found in typical variants. Two typical MCLs showed an anomalous single-stranded conformation polymorphism corresponding to the known polymorphisms at codon 148 of p16INK4a gene and codon 31 of p21Waf1 gene. These findings indicate that p21Waf1 and p16INK4a alterations are rare in typical MCLs but the loss of p21Waf1 and p16INK4a expression, and deletions of p16INK4a gene are associated with aggressive variants of MCLs, and they occur in a subset of tumors with a wild-type p53 gene.

Description: Abstract 4408 Chronic transfusion dependence increases the risk of developing iron overload which potentially could have a negative impact on the patient. It has not yet been studied in depth the profile of chronic transfusion-dependent patients, and their therapeutic management. Our objective was to describe the clinical characteristics of chronic transfusion-dependent patients in Spain. An epidemiologic, cross-sectional and multicenter study was performed in 41 Haematology Services according to clinical practice. Patients ≥18 years old, who started to be chronically transfused after January 2007 and who had received at least 10 red blood cell (RBC) units at the time of inclusion were enrolled in the study. A total of 631 patients were analyzed, 56.2% were men with mean age of 65.0 years (deviation standard=17.0). The principal cause of transfusion dependence was of haematological nature (92.9%), being myelodysplasic syndromes (35.8%) the most frequent cause, followed by acute myeloid leukaemia (28.8%), medullary aplasia (6.3%) and myeloproliferative syndromes (6.0%). 15.5% of patients with non-haematological causes of transfusion dependence was observed, being neoplasia the most frequent cause (6.7%). A median of 22 RBC units were transfused in total to the population in study and a median of 14 RBC units were transfused in the last year. 59.3% of patients received ≥20 RBC units. Regarding iron overload, median serum ferritin (SF) level was 1,173.5 ng/ml and 48.7% of patients had SF ≥1000 ng/ml. SF level was not available in 16.3% of patients. Moreover, 18.7% of patients had their last SF measurement done before the study initiation while 64.8% of the SF measurements were performed during the study. The concomitant diseases reported were mainly cardiovascular (24.4%), urogenital (11.7%) and endocrine (10.1%). A higher number of comorbidities was observed in the group of patients with SF ≥1000ng/ml, specially cardiovascular, hepatic and endocrine disease. Of all patients with chronic transfusion dependence, 14.1% received chelation therapy, being deferasirox the most commonly used drug (89.0%). The reason for not receiving chelation treatment was unknown in 28.2% of patients. 13.2% of patients with iron overload did not receive chelation therapy due to present concomitant diseases: neoplasias (6.7%) and renal and urinary disorders (3.0%). A good correlation between transfused RBC units and SF levels was observed in the study (59.3% received ≥20 RBC units, and 48.7% had SF levels ≥1000ng/ml). It was noted that monitoring of iron overload by SF levels is not followed properly in patients with chronic transfusion dependence. 16.3% of patients did not have any SF measurement and in 18.7% of patients the last SF determination was prior to the study. These data may be related to the fact that 28.2% of patients did not receive chelation therapy due to unknown causes. Disclosures: No relevant conflicts of interest to declare.

Description: Introduction: Standard therapy for acute myeloid leukaemia (AML) generally includes intense induction with daunorubicin (D) on days 1-3 and cytarabine (A) on days 1-7, followed by consolidation should complete remission (CR) be achieved. Assessment of bone marrow morphology, including percentage of blasts, remains the standard approach to gauge treatment response, however more sensitive molecular based approaches are capable of detecting subclinical levels of leukemic blasts (minimal residual disease, MRD). MRD often remains during and after standard treatment and is the main cause of relapse, a major problem in the management of AML. Resistance of the residual blasts to treatment can be attributed to the activity of pro-survival enzymes, some of which can be pharmacologically inhibited, however, finding the right inhibitor for the right patient presents a major challenge due to the plethora of different enzymes and combinations thereof. Liquid chromatography - tandem mass spectrometry (LC-MS/MS) proteomics enables global and unbiased quantification of protein expression and enzymatic activity in samples. We applied this technology to AML blasts at relapse compared to diagnosis, and in cell lines treated with standard chemotherapy to detect modulated biochemical pathways that contribute to resistance. Thorough investigation into the expression and activity of the protein drug targets enabled selection of inhibitors which proved effective when cells were treated in culture. This approach represents an effective way to better understand the biochemistry of cells following chemotherapy and identify suitable drug targets in biopsies to guide effective inhibitor selection. Methods: LC-MS/MS proteomics and phosphoproteomics was used to investigate global protein expression and kinase activity in primary AML samples at diagnosis and matched relapse (18 cases), and in 3 AML/APL cell lines before and after chemotherapy. Briefly, we collected frozen biopsy specimens from the Barts tissue bank and after thawing the AML blasts were incubated in media for 2 hr at 37oC. Cell lines (HL60, MV411 and P31/FUJ) were treated ± D and/or A (2, 6, or 24 hr). After incubation, cells were centrifuged and washed in PBS, then proteins extracted in urea lysis buffer. Proteins were digested with trypsin, and resulting peptides analysed directly by LC-MS/MS for proteomics or subjected to phosphopeptide enrichment using TiO2 for phosphoproteomics. Commercial (Mascot) and in-house (Pescal, KSEA) software were utilised to identify and quantify proteins, determine kinase activities and investigate intracellular signalling. Cell Viability of blasts ± treatments were recorded using the Guava ViaCount Reagent and Cytometer. Results: On average, 〉3000 proteins and 〉9,000 phosphorylation sites were identified per sample. One of the drug targets that correlated strongest with % blasts was CD99 (r=0.79). Blasts showed high abundance & activity of enzymes involved in DNA repair (e.g. PARP1, ATR and PRKDC) at diagnosis and relapse, several significantly increasing in relapse (e.g. PLK3 and APEX1). We observed significant increase in phosphorylation of signalling proteins, such as KIT and STAT5, in relapse. Other signalling pathways regulating survival, apoptosis and metabolism were modulated after relapse but these were patient specific. AML cell lines were more sensitive to D than A. HL60 was the most sensitive cell line while P31/FUJ were least sensitive. Chemotherapy significantly increased the activity of ATM, ATR, PRKDC and MAPKAPK2. Phosphorylation of HSPB1 increased significantly in the presence of D and/or A, and inversely correlated with sensitivity of cells to these drugs. Simultaneous inhibition of ATM & ATR significantly reduced P31/FUJ & MV411 cell viability ± A, while MAPKAPK2 inhibition increased sensitivity of MV411 cells to A. Conclusion: We identified the most abundant and active protein drug targets in AML primary samples and cell lines. Investigating primary AML at diagnosis and relapse uncovered changes in biochemical pathways that regulate DNA repair, survival, apoptosis and metabolism, some of them being modulated by chemotherapy in AML cell lines. These changes were often patient specific, suggesting that to effectively implement targeted therapies, a personalised approach is required and we demonstrate drug selection can be directed by LC-MS/MS proteomics. Disclosures No relevant conflicts of interest to declare.

Description: The molecular mechanisms underlying the pathogenesis of aggressive lymphomas and the histological transformation of indolent variants are not well known. To determine the role of p16INK4a gene alterations in the pathogenesis of non-Hodgkin's lymphomas (NHLs) and the histological progression of indolent variants, we have analyzed the expression, deletions, and mutations of this gene in a series of 112 NHLs. Hypermethylation of the gene was also examined in a subset of tumors with lack of protein expression but without mutations or deletions of the gene. p16INK4a gene alterations were detected in 3 out of 64 (5%) indolent lymphomas but in 16 out of 48 (33%) primary or transformed aggressive variants. In the low-grade tumors, p16INK4a alterations were detected in 1 (4%) chronic lymphocytic leukemia (hemizygous missense mutation), 1 (6%) follicular lymphoma (homozygous deletion), and 1 (5%) typical mantle cell lymphoma (homozygous deletion). The two later cases followed an aggressive clinical evolution. In the aggressive tumors, p16INK4a gene alterations were observed in 2 (29%) Richter's syndromes (2 homozygous deletions), 3 (33%) transformed follicular lymphomas (1 homozygous deletion and 2 nonsense mutations), 3 (43%) blastoid mantle cell lymphomas (2 homozygous and 1 hemizygous deletions), 5 (28%) de novo large-cell lymphomas (1 homozygous deletion and 4 hypermethylations), 2 lymphoblastic lymphomas (2 homozygous deletions), and 1 of 2 anaplastic large cell lymphomas (hypermethylation). Protein expression was lost in all tumors with p16INK4a alterations except in the typical chronic lymphocytic leukemia (CLL) with hemizygous point mutation. Sequential samples of the indolent and transformed phase of three cases showed the presence of p16INK4a deletions in the Richter's syndrome but not in the CLL component of two cases, whereas in a follicular lymphoma the deletion was present in both the follicular tumor and in the diffuse large-cell lymphoma. In conclusion, these findings indicate that p16INK4a gene alterations are a relatively infrequent phenomenon in NHLs. However, deletions, mutations, and hypermethylation of the gene with loss of protein expression are associated with aggressive tumors and they may also participate in the histological progression of indolent lymphomas.

Description: Mantle cell lymphoma (MCL) is molecularly characterized by bcl-1 rearrangement and cyclin D1 gene overexpression. Some aggressive variants of MCL have been described with blastic or large cell morphology, higher proliferative activity, and shorter survival. The cyclin-dependent kinase inhibitors (CDKIs) p21Waf1 and p16INK4a have been suggested as candidates for tumor-suppressor genes. To determine the role of p21Waf1 and p16INK4a gene alterations in MCLs, we examined the expression, deletions, and mutations of these genes in a series of 24 MCLs, 18 typical, and 6 aggressive variants. Loss of expression and/or deletions of p21Waf1 and p16INK4a genes were detected in 4 (67%) aggressive MCLs but in none of the typical variants. Two aggressive MCLs showed a loss of p16INK4a expression. These cases showed homozygous deletions of p16INK4a gene by Southern blot analysis. An additional aggressive MCL in which expression could not be examined showed a hemizygous 9p12 deletion. Loss of p21Waf1 expression at both protein and mRNA levels was detected in an additional aggressive MCL. No p21Waf1 gene deletions or mutations were found in this case. The p21Waf1 expression in MCLs was independent of p53 mutations. The two cases with p53 mutations showed p21Waf1 and p16INK4a expression whereas the 4 aggressive MCLs with p16INK4a and p21Waf1 gene alterations had a wild-type p53. p21Waf1 and p16INK4a were expressed at mRNA and protein levels in all typical MCLs examined. No gene deletions or point mutations were found in typical variants. Two typical MCLs showed an anomalous single-stranded conformation polymorphism corresponding to the known polymorphisms at codon 148 of p16INK4a gene and codon 31 of p21Waf1 gene. These findings indicate that p21Waf1 and p16INK4a alterations are rare in typical MCLs but the loss of p21Waf1 and p16INK4a expression, and deletions of p16INK4a gene are associated with aggressive variants of MCLs, and they occur in a subset of tumors with a wild-type p53 gene.

Description: We have prospectively evaluated the feasibility and results of the biotin-avidin immunoadsorption method (Ceprate SC system) for a phase I/II study of T-cell depletion of granulocyte colony-stimulating factor (G-CSF ) mobilized peripheral blood progenitor cells (PBPC) for allogeneic transplantation. Twenty consecutive patients, median age, 40 years (21 to 54) and diagnoses of chronic myeloid leukemia in chronic phase (n = 5), acute myeloblastic leukemia (n = 7), acute lymphoblastic leukemia (n = 2), chronic myelomonocytic leukemia (n = 1), refractory anemia with excess of blasts in transformation (n = 3), histiocytosis X (n = 1), and chronic lymphocytic leukemia (n = 1), were conditioned with cyclophosphamide (120 mg/kg) and total body irradiation (13 Gy; 4 fractions). HLA identical sibling donors received G-CSF at 10 μg/kg/d subcutaneously (SC); on days 5 and 6 (19 cases) and days 5 to 8 (1 case) donors underwent 10 L leukapheresis. PBPC were purified by positive selection of CD34+ cells using immunoadsorption biotin-avidin method (Ceprate SC) and were infused in the patients as the sole source of progenitor cells. No growth factors were administered posttransplant. The median recovery of CD34+ cells after the procedure was of 65%. The median number of CD34+ cells infused in the patients was 2.9 (range, 1.5 to 8.6) × 106/kg. The median number of CD3+ cells administered was 0.42 × 106/kg (range, 0.1 to 2). All patients engrafted. Neutrophil counts

Description: Myelodysplastic Syndrome (MDS) comprises a group of heterogeneous hematological disorders with variable risk of leukemic evolution (LE) and short survival (SV). According to French-American-British (FAB) classification, MDS are divided into five morphological entities and the World Health Organization reclassified them eliminating Refractory Anemia (RA) with Excess of Blast in transformation (RAEBt) and Chronic Myelomonocytic Leukemia (CMML), among other changes. Around 40–60% of patients shows normal karyotype at diagnosis and, despite this finding which is associated with good prognosis, some of them show poor outcome. The aim of this study was to evaluate different variables and prognostics scoring systems in an overall population (OP) of normal karyotyped MDS patients and in the reduced population (RP) after eliminating RAEBt and CMML subgroups. This retrospective study was performed in a population of 152 MDS patients with normal karyotype at diagnosis distributed according to FAB into 59% RA/RA with ringed sideroblast, 23% RA with excess of blasts, 5% RAEBt and 13% CMML. The OP’s median age was 67 (20–89) years with a sex ratio (M/F) of 1.53. The median follow-up was 25.7 (1–266) months (m) and there were 31 (20%) events of LE and 65 (43%) related-MDS death during this period. The OP showed a median SV of 57 m and time to LE (25%) of 44 m vs. 63 and 124 m, respectively for the RP. Age, sex, percentage of bone marrow (BM) blast, hemoglobin (Hb) level, platelets count and number of cytopenias were significant predictive variables for prognosis in both populations (Kaplan-Meier and Long-Rank test, p130, 39 y 21 m for the RP, respectively. Results showed that SV and LE risk should be evaluated separately in patients with normal karyotype using different variables to predict each one, at least in MDS-FAB classified patients or in those that present less than 20% of BM blasts and monocite counts lower than 1000/μL. These results would be helpful to develop better risk-adapted therapeutic strategies for MDS patients with normal karyotype.