bluuebunny/crossref_metadata_2025_split huggingface dataset:
POTENTIAL ROLE of 8-COLOR FLOW CYTOMETRY for CHARACTERIZATION of MDS with Del(5q) ABNORMALITIES
Authors: Oelschlaegel Uta , Sockel Katja , Mohr Brigitte , Thiede Christian , Ehninger Gerhard , Platzbecker Uwe 
Published: October 2019

Abstract
Abstract 5020
Four-colour based flow cytometry (FCM) of bone marrow (BM) samples has become a valuable tool in the diagnostics of patients (pts) with myelodysplastic syndrome (MDS). It is still unknown, whether aberrations detected by FCM differ between certain cytogenetic subgroups. By using an 8-color (5-tube) FCM diagnostic panel we aimed to investigate the antigen patterns of MDS pts with del(5q) abnormalities and compared these to MDS pts with non-del(5q) MDS.
BM samples of healthy donors (n=19) and 57 MDS pts including 28 pts (15 RCMD; 3 RAEB-1; 8 RAEB-2; 1 RAEB-t; 1 CMMoL-1) with del(5q) MDS (single n=15) and 29 pts (1 RCUD; 2 RA±RS; 17 RCMD; 2 RAEB-2; 7 sAML including 1 RAEB-t) with non-del(5q) MDS were investigated. FCM procedure was performed at a FACS Canto II (200,000 events) and standardized including the analysis of all samples after overnight storage at room temperature and erythrocytes lysis prior to antibody staining. Thresholds were set according to median ± 2SD and/or ½ log differences compared to healthy BM. Aberrations were scored according to the FCSS by Wells et al. 2003 and modified by Van de Loosdrecht et al. 2008.
In del(5q) MDS several phenotypic characteristics differed to non-del(5q) pts. Of note, lineage infidelity (CD2, CD5, CD7, CD19, CD56) was detectable in significantly fewer del(5q) pts compared to non-del(5q) MDS (in granulocytes: 8% vs. 33%, p=0.040 and in monocytes: 24% vs. 56%, p=0.027). Thus, especially aberrant CD56 expression contributed to these results with only small subpopulations being positive in del(5q) vs. often more than 50% of aberrant granulocytes/monocytes in non-del(5q) MDS pts. Additionally, the lymphoid-to-granulocytes/monocytes-ratios were higher in del(5q) (p=0.029 and 0.025) and hypogranularity was more pronounced in those pts (p=0.05). Intensity of expression of CD14 on monocytes as well as CD71 on granulocytes showed significant differences (2478 vs. 4402, p=0.035 and 2098 vs. 1552, p=0.007). Most of the above mentioned differences could also be confirmed if only RCMD pts of both groups were considered (e.g. lineage infidelity in granulocytes: 0% vs. 36%, p=0.041). Furthermore, trends were seen for a lower CD45-lymphocytes-to-blasts-ratio (4.8 vs. 5.4; p=0.09), higher CD33 intensity on blasts (2416 vs. 1673, p=0.08), and higher qualitative CD36 expression on granulocytes (3.2% vs. 2.5%, p=0.0619) as well as quantitative expression on monocytes (1940 vs. 663, p=0.06). Finally, monitoring of aberrations by FCM allowed for minimal residual disease monitoring in del(5q) MDS pts. Interestingly, the decreased CD19 expression on blasts seemed to be stable independent from the extent of treatment response.
Eight-colour based FCM is a valuable tool in the diagnostics of MDS. It might also be able to distinguish del(5q) MDS from other MDS subtypes.

Disclosures:
No relevant conflicts of interest to declare.
The Prognostic Implications of Nucleated Red Blood Cells in Myelodysplastic Syndrome
Authors: Munoz Javier , Younes Mohamad A. , Hammoud Marwa , Khanshour Ammar , Ortega Rosa Michel , Alhyari Mohammad , Yaghmour George , Kuriakose Philip 
Published: October 2019

Abstract
Abstract 5058

Background:
The presence of elevated nucleated red blood cells (NRBC) in the peripheral blood smear points towards the presence of myelodysplastic syndrome (MDS) or myelophthisis. The intent of our study was to assess the prognostic implication of the presence of increased NRBC in patients with MDS.

Methods:
Retrospective single institution chart review over a period of 10 years from 2000 to 2010. Our study population comprised 288 diagnosed cases of MDS whose diagnoses were made by bone marrow (BM) examination.

Results:
174 patients were male (60.4%) and 114 were female (39.6%). Average age at diagnosis of MDS was 71.92 years. 204 patients were Caucasian (70.8%), 69 patients were African-American (23.9%), 1 patient was Hispanic, 2 patients were Asian, and race was not documented in 12 patients. 117 patients had intermediate-1 IPSS (40.6%), 79 had low IPSS (27.4%), 54 patients had intermediate-2 IPSS (18.7%), 26 patients had high IPSS (9.0%), and 12 patients had unknown IPSS. 131 patients (45.5%) had undetectable NRBC in their peripheral smear prior to their initial diagnostic BM examination. 13 out of these 131 patients (9.92%) progressed to acute myeloid leukemia (AML). 157 patients (54.5%) had one or more NRBC in their peripheral smear prior to their diagnostic BM examination. 27 out of these 157 patients (17.19%) progressed to AML. Survival in days was available in 59 patients. 19 out of 59 patients had undetectable NRBC in their peripheral smear prior to their initial diagnostic BM examination with an average survival of 1,256.2 days. Only one patient in this group underwent allogeneic matched unrelated peripheral stem cell transplantation (PSCT) with a survival of 2,532 days. 40 out of 59 patients had one or more NRBC in their peripheral smear prior to their diagnostic BM examination with an average survival of 850.4 days. Only 3 patients in this group underwent allogeneic matched PSCT (2 related and 1 unrelated) with an average survival of 924.3 days.

Conclusions:
The presence of nucleated red blood cells in the peripheral blood smear of patients with MDS seems to be a marker of more aggressive disease, higher rates of progression to AML and portends worse survival. To our knowledge this is the first study documenting the prognostic implication of NRBC in the peripheral circulation of patients with MDS.

Disclosures:
No relevant conflicts of interest to declare.
4-1BBL Reverse Signaling Enhances Proliferation of Human Multiple Myeloma Cell Line U266
Authors: Wang Juan , Ju Songguang , Fu Chengcheng , Depei Wu 
Published: October 2019

Abstract
Abstract 5074
Multiple myeloma is an incurable plasma cell malignancy. Unraveling the intricate pathobiology and exploitation of novel therapeutic approaches are needed. 4-1BBL, a member of the tumor necrosis factor family, cross-links its receptor, 4-1BB, and co-stimulates T cell activities. Recent studies confirm that 4-1BB-4-1BBL interaction generates bi-directional signals. 4-1BBL has been detected on many tumor cells, and many tumor microenvironmental cells express 4-1BB molecule. However, whether the reverse signaling through 4-1BBL participates the pathology of multiple myeloma is unknown. In this study, we identify the constitutive expression of 4-1BBL on U266, LP1, MY5 and OPM2 MM cell lines as 89.2%, 57.9%, 77.9% and 78.7%. Then we select U266 cell to explore the biological effect of 4-1BBL reverse signaling and its underlying mechanism. Triggered by anti-4-1BBL mAb (1F1), cell counting and MTT assay both demonstrated that U266 cell proliferation increased dramatically. Intracellular cytokine staining showed that treatment of cells with 1F1 also increased the production of IL-6. When neutralizing anti-IL-6 mAb was added to the culture medium, 1F1-induced U266 cell proliferation was effectively inhibited. These effects of 4-1BBL require immobilization of 1F1 but not soluble form. Taken together, 4-1BBL reverse signaling promotes U266 cell proliferation remarkably while increasing the production of IL-6. Moreover, neutralizing anti-IL-6 mAb inhibits U266 cell proliferation stimulated by mAb 1F1, supporting that 4-1BBL reverse signaling promotes the proliferation of U266 cell through increasing the production of IL-6.

Disclosures:
No relevant conflicts of interest to declare.
Epigenetic Dysregulation of the Tissue Inhibitor of Metalloproteinases-2 Gene in Multiple Myeloma
Authors: Galm Oliver , Jost Edgar , Schubert Claudia , Herman James G. , Brümmendorf Tim H , Wilop Stefan 
Published: October 2019

Abstract
Abstract 5089

Background:
Multiple myeloma (MM) is a B-cell neoplasm characterized by the accumulation of malignant plasma cells in the bone marrow (BM) as well as abundant BM angiogenesis. Aberrant methylation of CpG islands near gene promoter regions is the most widely studied epigenetic abnormality in human malignancies and is associated with loss of gene expression. There is increasing evidence for a role of the tissue inhibitor of metalloproteinases 2 (TIMP2) gene as a tumor suppressor. Overexpression of TIMP2 may result in decreased invasive potential, suppression of tumor growth and vascularization as well as inhibition of angiogenesis. We could previously show that TIMP2 may become hypermethylated in association with transcriptional silencing in indolent and aggressive non-Hodgkin's lymphomas. Recently, downregulation of TIMP2 was also reported in MM, and this is thought to contribute to increased BM angiogenesis. In this study, we determined the methylation status of the promoter-associated CpG island of TIMP2 in primary MM samples and investigated correlations between TIMP2 methylation and clinical parameters.

Methods:
Methylation of the promoter-associated CpG island of TIMP2 was analyzed by methylation-specific polymerase chain reaction (MSP) in samples from 76 MM patients (median age 65 years [range 40–94]; 44 males, 32 females; 71 BM samples, 5 peripheral blood samples). Overall survival curves were plotted according to the method of Kaplan and Meier and compared using the log-rank test. Correlations between variables were analyzed using the Fisher's exact two-sided test and independent t-test, respectively.

Results:
MSP analysis revealed that there was aberrant methylation of the TIMP2 promoter region in 4/76 MM patient samples. We found an association of TIMP2 hypermethylation with plasma cell leukemia (p<0.0001) and decreased platelet counts (257.1 G/l vs. 111.5 G/l; p=0.04). There was a trend towards an inferior overall survival in MM cases with a hypermethylated TIMP2 gene, but this difference was not statistically significant (p=0.11).

Conclusions:
Our study shows that promoter hypermethylation of TIMP2 is a novel epigenetic event in MM that may contribute to disease progression and could serve as a prognostic biomarker. Further studies are warranted to elucidate the functional consequences of epigenetic dysregulation of TIMP2 in the pathogenesis of MM. Additionally, the increasing evidence for the role of DNA methylation changes in MM may serve as a basis for the use of epigenetically targeted therapeutic approaches in malignant plasma cell disorders.

Disclosures:
Herman: MDxHealth: Consultancy.
Lenalidomide Plus Bortezomib and Dexamethasone (RVD) Followed by Lenalidomide Plus Dexamethasone As Salvage Treatment for Myeloma Patients in First Relapse After Autologous Stem Cell Transplantation. Single Institution Experience
Authors: Belhadj Karim , Amraoui Kamel , Safar Violaine , Gaillard Isabelle , El Gnaoui Taoufik , Dupuis Jehan , Luciani Alain , Molinier-Frenkel Valérie , Rahmouni Alain , Haioun Corinne 
Published: October 2019

Abstract
Abstract 5143
The RVD regimen has shown a promising activity in patients with relapsed/refractory multiple myeloma (MM). In the present study, we report preliminary results on 14 patients in first relapse after autologous stem cell transplantation (ASCT) treated by VRD followed by a maintenance treatment with revlimid-dexamethasone (Rev-dex) in responder-patients.
Treatment was as follows: RVD included lenalidomide 15 mg per day (d) d1 through d14, bortezomib 1 mg/m2 on d1,4,8 and 11 and dexamethasone 20 mg on d1,2,4,5,8,9,11 and 12 of 21-d cycles with the objective to deliver at least 6 cycles. A maintenance treatment by Rev-dex with lenalidomide 25 mg d1 to d21 plus dexamethasone 20 mg weekly of d-28 cycles, delivered until progression was planned in patients achieving at least partial response to RVD.
Patients' characteristics at relapse: median age was 65 years (range 59 to 70), median time to relapse following ASCT before RVD was 26 months (range 4 to 86). All pts were naive for lenalinomide and 8 for bortezomib. Translocation t(4;14) was observed in 4 patients and was associated with del(17p13) in one case.
Patients received a median number of 6 cycles of RVD (range 3 to 8). Overall response rate was 71% and 43% of the patients achieved very good partial or better response. Discontinuation of RVD was due to disease progression in 4 cases. During RVD treatment, 4 patients (29%) experienced grade 3–4 adverse events: neutropenia n =2, thrombocytopenia n=1, thrombosis n=1. Following RVD, 10 patients received Rev-dex maintenance.
With a median follow-up of 15 months (range 6 to 29), 11 patients are alive free of evolutive disease. Four patients progressed during VRD: one with solitary plasmocytoma was efficiently treated by radiotherapy, the 3 other patients received anthracycline or platin based regimens but were refractory and 2 of them died. One patient progressed during maintenance and was efficiently treated by melphalan-cyclophosphamide-dexamethasone. Among the 4 patients with high-risk cytogenetic abnormalities, one CR, one PR and 2 progressions were observed during RVD. The first patient remains in CR 16 months after the beginning of salvage treatment.

Conclusion:
RVD followed by Rev-dex maintenance is a promising therapeutic option in MM patients in first relapse after ASCT with low toxicity profile.

Disclosures:
No relevant conflicts of interest to declare.
Incidence of the JAK2V617F Mutation In Mexican Patients with Myeloproliferative Neoplasms (MPNs)
Authors: Ignacio Gregorio , Arana-Trejo Rosa María , Gónzalez Verónica , Hérnandez Maria Paula , Lugo Yolanda , Solis Luis , Castellanos Jesús , Celestino Samuel , Váldez Carlos , Rivas Ramón 
Published: October 2019

Abstract
Abstract 5180

Introduction:
The V617F mutation in JAK2 gene has been described in approximately 50–90% of patients with ET, MF AND PV [essential trombocythaemia, idiopathic myelofibrosis and policithemia vera]; but has also been reported, albeit at a lower frequency, in patients with other myeloid malignancies such as atypical CML, CMML, AML, MDS, JMML and CNL. A single G>T base substitution in exon 12 results in the conversion of valine to a phenylalanine aminoacid at position 617 of the JAK2 gene. The identification in this study were using techniques such as allele-specific PCR, RFLP-PCR and direct sequencing; for to determine the incidence in Mexican patients with MPNs.

Patients and Methods:
The JAK2V617F mutation was determined in 88 patients and 5 normal blood samples for healthy individuals as controls. About the patients, 60 were cataloged like MPNs and 28 patients with features suggestive of MPNs vs CML. Samples for bone marrow or peripherical blood were taken either at time of diagnosis of MPNs or during treatment with cytoreductive or anti-thrombotic agents. DNA and RNA were extracted using the QIAamp DNA and RNeasy mini kit (Qiagen) and amplified by the three techniques mentioned for JAK2V617F and by nested RT-PCR for BCR/ABL.

Results:
The five normal blood samples for controls were negative for JAK2V617F mutation and to BCR/ABL. Patients had median age 65 years (47–85 years old), 46% male and 54% female. In de overall patients: 60 patients with MPNs all were BCR/ABL negative and 20 (33%) had JAK2V617F. In the 28 patients with likely MPNs vs CML, 23 were BCR/ABL positive/JAK2 negative, two had the coexistence of both genetics defects [BCR/ABL+ and JAK2V617F+] and 3 BCR/ABL and JAK2 negative. Finally the patiens with JAK2V617F+, were 12 ET, PV 1, MF 2, CML 2, and 5 continued like MPNs.

Discussion:
The incidence of the JAK2V617F in this study for MPNs patiens were 33% and the incidence varied between MPNs subtype. Less than ten cases of BCR/ABL+ CML with JAK2V617F have been published; we report two patients with the coexistence and we agree with previous reports that screening for JAK2V617F mutation should be considered in any BCR/ABL+ CML patients and the clinical outcome will be define in long period.

Disclosures:
No relevant conflicts of interest to declare.
Orbital Non Hodgkin Lymphomas. Features and Evolution of 63 Patients From Two Oncological Centers in Buenos Aires, Argentina: Marie Curie Hospital and Henry Moore Institute
Authors: Dragosky Marta O , Marquez Marisa , Luchetta Graciela P , Alcaraz Susana , Annetta Isabel , Corzo Ariel , Jozami Cecilia , Gil Deza Eduardo , Zerga Marta 
Published: October 2019

Abstract
Abstract 5201
Orbital Non Hodgkin Lymphoma (NHL) represents 2 to 5% of extranodal NHL and 8 to 11% of orbital tumors. Published reports usually comprise a reduced number of patients.
Out of a total of 668 extranodal NHL treated in the period 1991–2010, the orbital locations accounted for 63 patients (10.6%).This high value is due to selected referral from ophthalmologic centers. The diagnosis was made by cytology or surgical biopsy and immunohistochemistry. Staging included CT or MRI of the orbit and brain and bone marrow biopsy.

Results:
Age range:17 to 86 years (mean 60), only 5 < 40 years. The ratio M/F: 1/0.9.Location:right orbit: 50%,left: 36%, bilateral: 14%. Conjunctiva: 44.5%, orbit soft tissue: 23.8%, eyelid: 22.2%, lachrymal gland: 9.5%.
Initial symptoms: orbital tumor: 47.6%, proptosis: 26.9%, ptosis: 11.1%, eyelid edema: 6.3%, diplopia: 4.7%, epiphora: 3.2%.
Histology subtypes: MALT: 50.8%, lymphocytic: 30.1% LDGCB: 6.3%, follicular 6.3%, lymphoplasmacytic: 4.7%, angiocentric T: 1.6%. Time to diagnosis was above 12 months in 63.5%.
Treatment in 49 patients (77.7%). Chemotherapy (CT) (CVP, CHOP, CLB) plus radiotherapy (RT) in 57,14 %, CT in 28.57%, radiotherapy: 12.24%, Rituximab:.28% (12.24% + QT, 2.04% alone).
Complete remission (CR) was obtained in 87.77% and PR in 12.23%. Relapse in 16 (32.65%), local homo-or contralateral 9 (56.25%), systemic 6 (37.50%), 1 (6.25%) in breast (MALT). Died in progression: 4 (25%). The 46,93% of patients had survivals of 5 to 10 years, and 12.5% 11 to 15 years.

Conclusions
The outstanding features are: presentation in 5* to 6* decade, low grade subtypes, initial symptoms: painless mass and proptosis, prevalent location in conjunctiva and eyelid. Is remarkable the delay until diagnosis, due to its low frequency and low specificity of the initial symptoms. The high rate of complete remission obtained with chemotherapy and radiotherapy in frontline and relapse and prolonged survival, are characteristics that determine the good prognosis of this location of extranodal NHL.

Disclosures:
No relevant conflicts of interest to declare.
Role of FDG-PET/CT in Detecting Lymphomatous Bone Marrow Involvement in Patients with Newly Diagnosed Diffuse Large B-Cell Lymphoma
Authors: Hong Junshik , Lee Yukyung , Kim Seog Gyun , Hwang Kyung Hoon , Park Soon Ho , Jeong Jihoon , Kim Kyung-Hee , Ahn Jeong Yeal , Park Sanghui , Park Jinny , Lee Jae Hoon 
Published: October 2019

Abstract
Abstract 5204

Backgrounds:
As a bone marrow bopsy (BMB) is a painful and invasive procedure with a restrictive reliability as only a limited area of the bone marrow (BM) can be evaluated, role of FDG-PET/CT to demonstrate lymphomatous BM involvement as an alternative or at least a complementary to BMB is an area of interest. Several previous studies exist but most of them included heterogeneous types of lymphomas with various treatments.

Patients and methods:
To evaluate the role of FDG-PET/CT in detecting BM involvement, pre-treatment bilateral BMBs and FDG-PET/CT scans of 89 patients with diffuse large B-cell lymphoma (DLBCL) treated with standard immunochemotherapy, rituximab-CHOP were reviewed and analyzed. Uptake more than liver parenchyma intensity on FDG-PET/CT was interpreted as 'with a possibility' of involvement. The final interpretation on the possibility of BM involvement in each patient was reported after discussion among three nuclear medicine physicians and results of BMB were blinded at the time of FDG-PET/CT review. Fourteen patients (15.7%) had lymphomatous involvement based on BMB (BMB+) and 17 patients (19.1%) had the possibility of BM involvement on FDG-PET/CT (FDG-PET/CT+). Seventy-two patients (80.8%) had concordant results between BMB and FDG-PET/CT (7 patients were positive for both and 65 patients were negative for both), but 17 patients (19.2%) had a discordant interpretation (7 patients were BMB+ and FDG-PET/CT-, and 10 for BMB- and FDG-PET/CT+; table 1). Although BMB+ patients had an inferior 2-year EFS (37.0% vs. 79.8%, p < 0.001) and OS (36.3% vs. 81.0%, p < 0.001) compared to BMB- patients, no differences in EFS (62.6% vs. 72.7%, p = 0.185) and OS (59.4% vs. 78.0%, p = 0.146) were shown between FDG-PET/CT+ and FDG-PET/CT- patients. Six of 7 patients with BMB+ and FDG-PET/CT+ had a diffuse involvement on FDG-PET/CT whereas 9 of 10 patients with BMB- and FDG-PET/CT+ had a focal BM involvement on FDG-PET/CT (table 2). Six of 7 patients with diffuse involvement on FDG-PET/CT were BMB+ whereas only 1 of 10 patients with focal BM involvement on FDG-PET/CT were BMB+ (table 2). It is likely therefore that patients with diffuse BM involvement on FDG-PET/CT had higher probability for BMB+ and they might have poorer survival than those with focal BM involvement.

Conclusion:
The results suggest that FDG-PET/CT had a limited value to detect BM involvement in patients with DLBCL. It may not be justified to upgrade patient's Ann Arbor stage to IV according to focal hypermetabolic BM lesion on FDG-PET/CT. Until additional results on the role of FDG-PET/CT in detecting BM involvement available, FDG-PET/CT should be used as an adjuvant rather than an alternative in detecting BM involvement in patients with newly diagnosed DLBCL.

Disclosures:
No relevant conflicts of interest to declare.
Gemcitabine, L-Asparaginase, Ifosfamide, Dexamethasone and Etoposide (GLIDE) Regiment In Advanced or Refractory Aggressive NK/T Cell Lymphomas: A Pilot Study
Authors: Ji Jie , Liu Ting , Xiang Bing , Jia Yongqian 
Published: October 2019

Abstract
Abstract 5208

Background:
Extra nodal NK/T cell lymphoma, nasal type (ENKL) with advanced stage and aggressive NK-cell leukemia (ANKL) are highly aggressive neoplasm with a dismal clinical outcome. Both of these diseases presented with rapid progressed clinical course and resistant to chemotherapy regimens such as CHOP(cyclophosphamide, doxorubicin, vincristine and prednisone). Expression of multi-drug resistance (MDR) associated P-glycoprotein of NK/T cell neoplasms which actively exports many anti-tumor agents outside the tumor cells is one of the well-studied mechanisms. Our new chemotherapeutic regimen GLIDE (Gemcitabine, L-asparaginase, Isofosfomide, Dexamethasone and Epotoside) showed promising results.

Patients and methods:
From 2010–2011, 3 cases of newly diagnosed ENKL,1 case of newly diagnosed ANKL, and 3 cases of refractory ENKL were enrolled. Former treatments included at least 2–6 cycles of chemotherapy (CHOP or L-asparaginase containing regimens) and local radiotherapy. All of these patients received chemotherapy of GLIDE regimen(Gemcibabine 1000mg/m̂2 D1,8; L-asparaginase 6000u/m̂2 D4,6,8,10,12; Isofosfomaide 1000mg/m̂2 D1-3;Dexamethasone 20mg D1-4; Epotoside 100mg/m̂2 D1-3).The stage of all patients was IV. IPI scores were all in high risk. All patients provided informed written consents and this trial was registered at http://www.chictr.org (ChiCTR-TNC-10000782)

Results:
The median age was 41 years (range, 23–54 years);and male (n = 2) to female (n = 5) was 1:2.5. 4 patients finished all 6 cycles, with 3 in CR and 1 in PR; 1 patient died of progression of disease after 2 cycles; 1 patient died of infection in nCR and 1 patient just finished cycle 2 with PR. The OR (CR+nCR+PR) was 85%. The major toxicity of GLIDE were grade 3/4 leucopenia (70.1%, 22 of 31 cycles), and infection (36.3%, 12 of 31 cycles) with 1 death. The non-hematologic toxicity was tolerable, and the hepatic toxicity-associated with the use of L-asparaginase was frequent (42.9%, 3 of 7 patients). However, the majority of the hepatic toxicities were grade 1 or 2.

Conclusion:
GLIDE chemotherapy can be an effective treatment strategy with acceptable toxicity in advanced stage or refractory ENKL and ANKL.

Disclosures:
No relevant conflicts of interest to declare.