Japanese Journal of Clinical Oncology 31:30-34 (2001)
© 2001 Foundation for Promotion of Cancer Research
Possible Clinical Benefits of the Use of Peripheral Blood Stem Cells Over Bone Marrow in the Allogeneic Transplantation Setting for the Treatment of Childhood Leukemia
1Pediatric Oncology Division and 2Hematopoietic Stem Cell Transplantation Division, National Cancer Center Hospital, Tokyo, Japan
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Background: The benefits of allogeneic peripheral blood stem/progenitor cell transplantation (PBSCT) over bone marrow transplantation (BMT), if any, have not been seriously evaluated in a pediatric population. We report here our experience with this procedure and demonstrate rapid engraftment to reduce procedure-related complications and enhanced allogeneic immune reaction to reduce leukemic relapse.
Methods: The feasibility of PBSCT was reviewed retrospectively. Four patients (2 AML and 2 ALL, aged 8–18 years) underwent allogeneic PBSCT for relapsed leukemia after primary allogeneic BMT (n = 2), for active hepatosplenic fungal abscess (n = 1) or for refractory relapse with conventional chemotherapy (n = 1). Four healthy donors (aged 10–49 years) received granulocyte colony-stimulating factor (G-CSF) 10 µg/kg/day by subcutaneous injection for 5 days. An individualized cytoreductive regimen was used before transplantation.
Results: No significant toxicities were observed in normal donors on G-CSF treatment or at collection of PBSC. After PBSCT, no significant acute toxicities were observed and the median duration to an absolute granulocyte count of 0.5 x 109/l and a platelet count of 20 x 109/l was 16 and 21 days, respectively. Although none of our patients developed acute graft-versus-host disease (GVHD), two developed chronic GVHD involving the liver and skin. Among those who developed chronic GVHD, one died of recurrent disease and another died of pneumonia 235 days after PBSCT. The two remaining patients have been alive without evidence of disease with follow-ups of 193 and 123 days, respectively.
Conclusions: Allogeneic PBSCT can be a safe procedure in a pediatric population with fewer acute complications, although the potential risk of G-CSF treatment in normal donors should be seriously weighed against the existing risks of marrow aspiration under general anesthesia. The risk of chronic GVHD may need to be balanced against a possible graft-versus-leukemia benefit in patients at higher risk of leukemic relapse.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The hematopoietic stem cell transplantation procedure with cytokine-mobilized peripheral blood stem/progenitor cells (PBSC) was first developed in an autograft setting and then began to be applied in an allogeneic setting. Compared with conventional allogeneic bone marrow transplantation (BMT), PBSCT has practical advantages such as immediate hematopoietic reconstitution resulting in few infectious complications and possible enhancement of the graft-versus-leukemia (GVL) effect (1). However, there is limited information available regarding allogeneic PBSCT in a pediatric population (2–4), mainly because of the technical difficulties in collecting PBSC and the unresolved ethical question regarding the use of granulocyte colony-stimulating factor (G-CSF) in normal children (5,6). We report here our experience with allogeneic PBSCT for pediatric patients with acute leukemia who were transplanted from an HLA-matched related donor, which suggests that PBSCT may offer advantages over BMT.
|
PATIENTS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Patients
Four patients [two acute myelogenous leukemia (AML) and two acute lymphoblastic leukemia (ALL)] underwent allogeneic PBSCT between May 1999 and March 2000 at the National Cancer Center Hospital of Japan (NCCH). The patients’ characteristics are summarized in Table 1. The patients are numbered based on the date of PBSCT. All four patients were females and they ranged in age from 8 to 18 years (median, 10 years). Associated factors for a poor prognosis included relapse after allogeneic BMT (n = 2; cases 1 and 4), active hepatosplenic fungal abscess (case 2) and refractory relapse with conventional chemotherapy (case 3). Each patient and donor, and/or their parents, provided written informed consent and the protocol was approved by the institutional review board of NCCH.
|
PBSC Harvest
Four healthy donors (three males and one female) aged 10–49 years (median 21 years) received G-CSF (filgrastim) at a dose of 10 µg/kg/day by subcutaneous injection for 5 days. The details of this procedure have been published elsewhere (7,8). On days 5 and 6 of G-CSF treatment, donors underwent apheresis with a Cobe Spectra continuous cell separator (Cobe Laboratories, Denver, CO). Briefly, for withdrawal of blood, no central venous catheterization was used and apheresis was run through a 22-gage catheter placed in the radial artery or a 19–22-gage catheter in the cubital or femoral vein. Cannulation and collection were performed without sedation. Acid citrate dextrose (ACD) and 5000 IU of preservative-free heparin were infused as an anticoagulant with whole blood: anticoagulant in a ratio of 12:1 and calcium gluconate was administered during apheresis to prevent ACD toxicity. A volume of 85–240 ml per donor’s body weight of blood (maximum 10 l) was processed at a time. Collected cells were frozen and stored in liquid nitrogen at –196°C. For cell evaluation, cells were labeled with fluorescence-conjugated anti-CD34 (HPCA) and analyzed using a flow cytometer. Donors were followed up every 6 months for blood counts.
Transplant Procedure
A combination of busulfan (BU) 14 mg/kg + cyclophosphamide (CY) 120 mg/kg, total body irradiation (TBI) 12 Gy + cytarabine (CA) 12 g/m2 + CY 120 mg/kg, TBI 12 Gy + CA 12 g/m2 + melphalan (PAM) 180 mg/ m2 or TBI 12 Gy + etoposide (VP-16) 60 mg/kg + CY 120 mg/kg was administered as a preparative regimen (Table 1). Cryopreserved apheresis products were thawed and infused 24 h after the completion of conditioning. Cyclosporine (CsA) and a short course of methotrexate (MTX) were used for graft-versus-host disease (GVHD) prophylaxis. CsA was given at a dose of 3 mg/kg body weight by continuous intravenous infusion, starting on day –1. The dose was reduced after engraftment and then switched to oral medication. When GVHD was well controlled, CsA was gradually tapered. In cases 1, 2 and 3, CsA was tapered and stopped on days 36, 101 and 68, respectively, expecting induction of GVL effect associated with GVHD. Methotrexate was also given at 15 mg/m2 on day +1 and 10 mg/m2 on days 3, 6 and 11. Acute GVHD was graded continuously according to the Przepiorka criteria (9). Chronic GVHD was classified as described by Shulman et al. (10). Skin biopsy was performed as necessary.
All patients were nursed in a filtered-air-flow single room, but no gut decontamination was given. All of the patients received intravenous hyperalimentation and blood products when needed. All blood products were irradiated and filtered. Acyclovir was administered at 5 mg/kg intravenously every 8 h from day –7 to day +35. Intravenous immunoglobulin 70–150 mg/kg was given weekly until discharge from the hospital. Broad-spectrum antibiotics were given for fever associated with neutropenia as required. G-CSF was used in three patients to enhance granulocyte engraftment.
Engraftment Analysis
Granulocyte recovery was defined as the first of two consecutive days with an absolute granulocyte count (AGC) of >0.5 x 109/l and platelet recovery was defined as the day when the platelet count rose to >20 x 109/l without platelet transfusion support. Hematopoietic chimerism was evaluated by fluorescence in situ hybridization (FISH) in gender-mismatched patient–donor pairs and by short tandem repeats (STR) in gender-matched pairs.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Mobilization and Collection of PBSC
The toxicities related to G-CSF treatment in donors were acceptable. One donor complained of bone pain and arthralgia, which were easily relieved by analgesics. The procedure for PBSC harvest was well tolerated and no significant complications, including hemodynamic instability, were noted during the procedure. Two consecutive days of apheresis were needed to collect a sufficient number of PBSC in all donors. For drawing blood, a radial artery was used in one donor (10 years) and a cubital or femoral vein was used in the remaining three donors (10, 14 and 49 years). The blood flow rate ranged from 25 to 60 ml/min. During PBSC collection, no clinical evidence of hypocalcemia was observed. The platelet count decreased in all of the donors, but none needed re-infusion of their own platelet-rich plasma that had been obtained during cell processing. There was no clinically significant bleeding. Other blood tests showed no abnormal values in any of the donors after apheresis procedures.
Engraftment
The number of progenitor cells infused and engraftment results are shown in Table 1. The patients received a median of 7.1 (range 5.31–9.96) x 106/kg CD34 positive cells. The peripheral blood cell count recovered in all of the patients and the median number of days to an AGC of >0.5 x 109/l was 16 (range 13–21), and that to a platelet count of >20 x 109/l was 21 (11–30). At 1 month following PBSCT, FISH and STR studies showed complete chimera in all patients.
Complications and Outcomes
All four patients developed mild to moderate oropharyngeal pain associated with mucositis and three required narcotics to relieve the pain. One patient had elevated transaminase levels greater than five times normal. Hemorrhagic cystitis did not occur in any of the patients. All patients had a high temperature shortly after PBSCT. However, the fever subsided within 5 days in all patients with a combination of antibiotics. One patient (case 2) received intravenous liposomal amphotericin B to manage her baseline fungal infection.
Two (cases 1 and 2) of the four patients who were followed at least 100 days developed chronic GVHD. One patient (case 2) had limited chronic GVHD of the skin and liver and received immunosuppressive therapy consisting of CsA and steroids. The other patient (case 1) developed extensive chronic GVHD of the liver and lung. She developed bronchiolitis obliterans and restarted immunosuppressive therapy consisting of CsA and steroids. However, this patient died from progressive GVHD of the lung at day +235. Case 2 died from bacterial pneumonia on day +235 after relapse, which occurred at day +103. The remaining two patients have been alive without evidence of disease with follow-ups of +193 (case 3) and +123 days (case 4). Case reports of two characteristic patients (cases 2 and 4) are given below.
Case 2
A 9-year-old girl diagnosed with AML (FAB M2) in September 1998, who was complicated with systemic fungal infection with abscess formation in the liver and spleen in her treatment course, underwent allo-PBSCT from her HLA-identical father. Briefly, the WBC count at the diagnosis of leukemia was 8.2 x 109/l and cytogenetics of leukemia cells demonstrated an abnormal karyotype of 46 XX, t(6;9)(p23;q34). She was not able to achieve remission with several chemotherapy regimens such as CA, VP-16, mitoxantrone (MIT) and pirarubicin (THP). Low-dose chemotherapy with CA (20 mg/m2 x 18 days) and aclarubicin (20 mg/m2 x 2 days) administered with rhG-CSF (2 µg/kg x 18 days) was then incorporated as a second-line induction therapy. During this course of therapy, she became severely neutropenic and developed high-grade fever, which did not respond to intensive antibacterial treatment with broad-spectrum intravenous antibiotics including imipenem/cilastatin sodium (60 mg/kg/day). Chest radiographs and blood cultures failed to demonstrate any evidence of infectious etiology. Ultrasonography (US) and computed tomography of the abdomen showed multiple round low-echogenic and low-density areas in the liver and spleen and a diagnosis of deep fungal infection was made. After starting conventional amphotericin B (0.7 mg/kg/day), the fever subsided and the abscesses continued to decrease in size. Repeated US revealed no abscess in the liver or spleen after 2 months. In preparation for the scheduled allogeneic transplantation, plain amphotericin B was replaced with liposomal amphotericin B (2 mg/kg/day) to intensify the antifungal effect. She then underwent allo-PBSCT on relapse in the hope of inducing a GVL effect for the leukemia with induction failure and rapid engraftment for the control of fungal infection. The conditioning regimen consisted of CY 120 mg/kg, CA 12 g/m2 and 12 Gy of TBI in six fractions for 3 days. A total of 9.96 x 106/kg of CD34 positive cells that had been previously harvested from her HLA-identical father were infused following the conditioning. CsA (3 mg/kg) and a short course of MTX (15 mg/m2 on day 1, 10 mg/m2 on days 3, 6 and 11) were given as a GVHD prophylaxis. No G-CSF was administered after PBSCT. The AGC reached 0.5 x 109/l on day 21 after PBSCT. A platelet count of >20 x 109/l was achieved on day 23. After the engraftment, the dose of liposomal amphotericin B was gradually tapered off and no complication was observed in the early course of transplant. She was discharged from the hospital without any evidence of GVHD or relapse on day 74 after allo-PBSCT. However, on day 89 after PBSCT, her leukemia recurred in the bone marrow. With the expectation of a GVL effect, CsA for GVHD prophylaxis and steroid treatment for the probable engraftment syndrome were discontinued. Combination chemotherapy including CA and VP-16 started and donor lymphocyte infusion was performed on day 105. However, her disease continued to progress and resulted in death from bacterial pneumonia 235 days after transplantation.
Case 4
An 8-year-old girl with AML, who had recurrent disease 11 months after allogeneic BMT from her HLA-matched brother, underwent secondary allo-PBSCT from the same donor. She was primarily diagnosed with AML (FAB M2) in October 1996. The WBC count at diagnosis was 10.8 x 109/l and the karyotype of the leukemia cells was 46 XX, t(8;21)(q22;q32). Her first complete remission (CR) was achieved with induction chemotherapy including VP-16, CA and MIT. Eight courses of consolidation chemotherapy including VP-16, CA, MIT, THP, aclarubicin and vincristine were then administered and 18 Gy irradiation was administered to the whole brain. Eleven months after induction therapy, the leukemia relapsed and reinduction chemotherapy including VP-16, CA and MIT was administered and she achieved a second CR. In October 1997, she underwent allo-BMT. The conditioning regimen consisted of BU 16 mg/kg, VP-16 60 mg/kg and PAM 180 mg/m2. MTX alone (15 mg/m2 on day 1, 10 mg/m2 on days 3, 6 and 11 and then weekly until day 102) was given as prophylaxis against GVHD, but evidence of acute GVHD in the skin (grade II) and digestive tract (grade I) became apparent. The AGC increased to over 0.5 x 109/l 25 days after BMT. A platelet count of >20 x 109/l was achieved 17 days after BMT. Although chronic GVHD developed after acute GVHD, the leukemia relapsed again 10 months after allo-BMT. Donor lymphocyte infusion and reinduction chemotherapy including VP-16, CA and MIT were administered, but CR was not achieved. She received allo-PBSCT on relapse with a conditioning regimen of CY 120 mg/kg, CA 12 g/m2 and 12 Gy of TBI in six fractions for 3 days. The patient then received an infusion of PBSC containing 5.31 x 106/kg harvested CD34 positive cells from the same donor as in the first transplant. CsA (3 mg/kg) and a short course of MTX (15 mg/m2 on day 1, 10 mg/m2 on days 3, 6 and 11) were given as prophylaxis against GVHD. G-CSF was administered for 13 days after PBSCT. The AGC increased to over 0.5 x 109/l 16 days after PBSCT. A platelet count of >20 x 109/l was achieved 21 days after PBSCT. She developed mild oropharyngeal pain with mucositis, but no other toxicities including acute GVHD were observed. At 3 months after PBSCT, she achieved CR again. Her bone marrow showed donor-karyotype (46, XY) and FISH for STR showed complete chimera.
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
In adult series, allogeneic PBSCT resulted in rapid hematopoietic reconstitution without excessive acute GVHD compared with allogeneic BMT (11–13), resulting in fewer infectious complications and a shorter hospitalization (14). In our experience with a pediatric population, no significant toxicities were observed at collection of PBSC and all of the patients achieved reasonably fast engraftment after PBSCT without serious complications. The median number of days to an AGC of >0.5 x 109/l and a platelet count of >20 x 109/l were 16 and 21 days, respectively. Hence these results support the previously reported feasibility of allogeneic PBSCT in children (4,15,16). In this study, we evaluated whether rapid engraftment translated into fewer complications. Case 2, who was recently complicated with systemic fungal infection, could be safely managed during the PBSCT procedure.
The risk of chronic GVHD in allo-PBSCT is reportedly higher than that in conventional BMT (1). Studies in adult recipients of allo-PBSCT have demonstrated an increased risk of chronic GVHD in up to 80% of the recipients (1,17). Although two of our patients developed chronic GVHD involving the liver and skin, both episodes were induced with intentional early tapering of CsA expecting favorable GVL effect for recurrent leukemia. Therefore, it is uncertain whether the risk of chronic GVHD associated with allo-PBSCT is also high in pediatric patients. Among those who developed chronic GVHD, one had recurrent disease and died and another died of pneumonia 235 days after PBSCT. At least in these two patients, GVL effect was not adequate to prevent leukemic relapse after transplantation.
The other two patients received allo-PBSCT as a second stem cell transplant from the same donor as in the first BMT. Hence they were expected to have increased procedure-related toxicities and leukemic relapse. However, after allogeneic PBSCT, both recipients achieved rapid granulocyte engraftment without major complications and remain in continuous disease-free status to date, although the observation period has been short (18–20). Although these two patients did not develop apparent GVHD, the isolated GVL effect might have contributed to the good outcome. In addition, the patient with serious infectious complications (case 2) also tolerated allo-PBSCT well as described above. These data suggest that allo-PBSCT can be safely applied to patients who are compromised with serious complications and/or leukemic recurrence after first BMT. In conclusion, we suggest that allo-PBSCT is a safe and effective treatment option for patients with an increased risk of procedure-related complications and/or leukemic recurrence.
|
FOOTNOTES |
|---|
+ For reprints and all correspondence: Atsushi Makimoto, Pediatric Oncology Division, National Cancer Center Hospital, 1–1 Tsukiji 5-Chome, Chuo-ku, Tokyo 104-0045, Japan. E-mail: amakimot@ncc.go.jp
Abbreviations: PBSCT, peripheral blood stem cell transplantation; BMT, bone marrow transplantation; GVHD, graft-versus-host disease; GVL, graft-versus-leukemia; G-CSF, granulocyte colony-stimulating factor; NCCH, National Cancer Center Hospital of Japan; AML, acute myelogenous leukemia; ALL, acute lymphoblastic leukemia; ACD, acid citrate dextrose; BU, busulfan; CY, cyclophosphamide; TBI, total body irradiation; CA, cytarabine; PAM, melphalan; VP-16, etoposide; CsA, cyclosporine A; MTX, methotrexate; AGC, absolute granulocyte counts; FISH, fluorescence in situ hybridization; STR, short tandem repeats; MIT, mitoxantrone; THP, pirarubicin; US, ultrasonography; CR, complete remission; S, skin; Li, liver; Lu, lung
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONPATIENTS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
1 Champlin RE, Schmitz N, Horowitz MM, Chapuis B, Chopra R, Cornelissen JJ, et al. Blood stem cells compared with bone marrow as a source of hematopoietic cells for allogeneic transplantation. Blood 2000;95:3702–9.
2 Diaz MA, Alegre A, Villa M, Benito A, Bernardo MR, Lopez-Botet M, et al. Allogeneic peripheral blood progenitor cell (PBPC) transplantation in children with haematological malignancies. Br J Haematol 1997;96:161–4.[Web of Science][Medline]
3 Li CK, Yuen PM, Chik KW, Shing MM, Li K, Tsang KS, et al. Allogeneic peripheral blood stem cell transplant in children. Med Pediatr Oncol 1998;30:147–51.[Web of Science][Medline]
4 Watanabe T, Kajiume T, Abe T, Kawano Y, Iwai A, Iwai T, et al. Allogeneic peripheral blood stem cell transplantation in children with hematologic malignancies from HLA-matched siblings. Med Pediatr Oncol 2000;34:171–6.[Web of Science][Medline]
5 Nussbaumer W, Schonitzer D, Trieb T, Fink FM, Maurer-Dengg K, Hocker P, et al. Peripheral blood stem cell (PBSC) collection in extremely low-weight infants. Bone Marrow Transplant 1996;18:15–7.[Web of Science][Medline]
6 Galacki DM. An overview of therapeutic apheresis in pediatrics. J Clin Apheresis 1997;12:1–3.[Web of Science][Medline]
7 Takaue Y, Kawano Y, Abe T, Okamoto Y, Suzue T, Shimizu T, et al. Collection and transplantation of peripheral blood stem cells in very small children weighing 20 kg or less. Blood 1995;86:372–80.
8 Makimoto A, Kawano Y, Abe T, Okamoto Y, Sato J, Nakagawa R, et al. Comparative evaluation of procedures with a Baxter CS-3000 cell separator for collecting peripheral blood cells from children. J Hematother 1999;8:305–10.[Web of Science][Medline]
9 Przepiorka D, Weisdorf D, Martin P, Klingemann HG, Beatty P, Hows J, et al. 1994 Consensus Conference on Acute GVHD Grading. Bone Marrow Transplant 1995;15:825–8.[Web of Science][Medline]
10 Shulman HM, Sullivan KM, Weiden PL, McDonald GB, Striker GE, Sale GE, et al. Chronic graft-versus-host syndrome in man. A long-term clinicopathologic study of 20 Seattle patients. Am J Med 1980;69:204–17.[Web of Science][Medline]
11 Korbling M, Przepiorka D, Huh YO, Engel H, van Besien K, Giralt S, et al. Allogeneic blood stem cell transplantation for refractory leukemia and lymphoma: potential advantage of blood over marrow allografts. Blood 1995;85:1659–65.
12 Bensinger WI, Clift R, Martin P, Appelbaum FR, Demirer T, Gooley T, et al. Allogeneic peripheral blood stem cell transplantation in patients with advanced hematologic malignancies: a retrospective comparison with marrow transplantation. Blood 1996;88:2794–800.
13 Schmitz N, Bacigalupo A, Labopin M, Majolino I, Laporte JP, Brinch L, et al. Transplantation of peripheral blood progenitor cells from HLA-identical sibling donors. European Group for Blood and Marrow Transplantation (EBMT). Br J Haematol 1996;95:715–23.[Web of Science][Medline]
14 Pavletic ZS, Bishop MR, Tarantolo SR, Martin-Algarra S, Bierman PJ, Vose JM, et al. Hematopoietic recovery after allogeneic blood stem-cell transplantation compared with bone marrow transplantation in patients with hematologic malignancies. J Clin Oncol 1997;15:1608–16.[Abstract]
15 Miniero R, Busca A, Bonetti F, Giorgiani G, Zecca M, Berger M, et al. Allogeneic transplantation of peripheral blood progenitor cells in children: experience of two pediatric centers. Bone Marrow Transplant 1998;22(Suppl 5):S33–6.
16 Levine JE, Wiley J, Kletzel M, Yanik G, Hutchinson RJ, Koehler M, et al. Cytokine-mobilized allogeneic peripheral blood stem cell transplants in children result in rapid engraftment and a high incidence of chronic GVHD. Bone Marrow Transplant 2000;25:13–8.[Web of Science][Medline]
17 Storek J, Gooley T, Siadak M, Bensinger WI, Maloney DG, Chauncey TR, et al. Allogeneic peripheral blood stem cell transplantation may be associated with a high risk of chronic graft-versus-host disease. Blood 1997;90:4705–9.
18 Bosi A, Bacci S, Miniero R, Locatelli F, Laszlo D, Longo G, et al. Second allogeneic bone marrow transplantation in acute leukemia: a multicenter study from the Gruppo Italiano Trapianto di Midollo Osseo (GITMO). Leukemia 1997;11:420–4.[Web of Science][Medline]
19 Blau IW, Basara N, Bischoff M, Gunzelmann S, Romer E, Kirsten D, et al. Second allogeneic hematopoietic stem cell transplantation as treatment for leukemia relapsing following a first transplant. Bone Marrow Transplant 2000;25:41–5.[Web of Science][Medline]
20 Michallet M, Tanguy ML, Socie G, Thiebaut A, Belhabri A, Milpied N, et al. Second allogeneic haematopoietic stem cell transplantation in relapsed acute and chronic leukaemias for patients who underwent a first allogeneic bone marrow transplantation: a survey of the Société Française de Greffe de Moelle (SFGM). Br J Haematol 2000;108:400–7.[Web of Science][Medline]
Received August 21, 2000; accepted October 17, 2000.
CiteULike 
Connotea 
Del.icio.us What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||