CD34+/CD90+ cells infused best predict late ... - Wiley Online Library

British Journal of Haematology, 2002, 117, 238–244

CD34+/CD90+ cells infused best predict late haematopoietic reconstitution following autologous peripheral blood stem cell transplantation Toshiya Sumikuma, Chihiro Shimazaki, Tohru Inaba, Naoya Ochiai, Akira Okano, Mayumi Hatsuse, Eishi Ashihara and Masao Nakagawa Second Department of Medicine, Kyoto Prefectural University of Medicine, Kamigyoku, Kyoto, Japan Received 28 June 2001; accepted for publication 18 October 2001

Summary. This study aimed to identify which graft product subset of cells might be the most predictive of late haematopoietic recovery (three to 12 months) following autologous peripheral blood stem cell transplantation (PBSCT). The relationships between the numbers of reinfused CD34+ cells and their immature subsets such as CD34+/CD90+, CD34+/AC133+, CD34+/CD38– and CD34+/HLA-DR– cells, and haemoglobin, white blood cell (WBC) and platelet counts at 3, 6, 9 and 12 months after PBSCT, were studied in 25 patients with haematological and solid malignancies. The total CD34+ cell number, as well as CD34+/CD90+ and CD34+/AC133+ cell numbers, correlated with platelet counts at 3, 6, 9 and 12 months after PBSCT, but the

CD34+/CD90+ cells infused best predicted platelet recovery during the first 12 months after PBSCT (P < 0Æ0238 at any time-point). The CD34+/AC133+ cell dose also correlated with WBC counts at 3 months post PBSCT. In addition, all patients receiving more than 80 · 104 CD34+/CD90+ cells/kg showed platelet counts greater than 100 · 109/l at all points after PBSCT, suggesting that this value of the CD34+/CD90+ cells infused was a threshold dose for durable haematopoietic engraftment after PBSCT.

Autologous peripheral blood stem cell transplantation (PBSCT) has been increasingly used after high-dose chemotherapy for patients with haematological or non-haematological malignancies (Kessinger & Armitage, 1991; Shimazaki et al, 1992). It is necessary to infuse an adequate quantity of stem cells for stable haematopoietic reconstitution after PBSCT. Several studies have shown a correlation between the number of granulocyte-macrophage colonyforming units (CFU-GM) and CD34+ cells, and the time to granulocyte and platelet recovery (Siena et al, 1991; Bensinger et al, 1994; Weaver et al, 1995; Henon et al, 1998; Pecora et al, 1998). The number of committed progenitor cells expressing myeloid (CD33, CD13), erythroid (CD71) and megakaryocytic (CD41, CD61) markers, has also shown some correlation with early engraftment after PBSCT (Dercksen et al, 1995; Feng et al, 1998; Stewart et al, 1999). In addition, a few clinical studies have shown a

correlation between the number of CD34+ cells and longterm haematopoietic reconstitution (6–12 months) (Haas et al, 1995a; Kiss et al, 1997; Amigo et al, 1999; PerezSimon et al, 1999; Duggan et al, 2000). However, the relationships between the number of infused immature progenitors and the stem cell fraction and late haematopoietic reconstitution have not been extensively studied (Duggan et al, 2000). In the present study, we analysed the number of immature progenitor and stem cells infused using monoclonal antibodies CD90, AC133, CD38 and HLA-DR, and examined the relationship between the number of these cells and late haematopoietic reconstitution during the first 12 months after PBSCT.

Correspondence: Dr Chihiro Shimazaki, Second Department of Medicine, Kyoto Prefectural University of Medicine, 465 Kawaramachi-Hirokoji, Kamigyoku, Kyoto 602–8566, Japan. E-mail: [email protected]

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Keywords: autologous peripheral blood stem cell transplantation, CD34+ cells, CD90+ cells, AC133+ cells, late haematopoietic recovery.

MATERIALS AND METHODS Patients. A total of 25 patients with haematological and solid malignancies were included in this study; six with acute myelogenous leukaemia (AML), one with acute lymphoblastic leukaemia (ALL), 13 with non-Hodgkin’s lymphoma (NHL), one with Hodgkin’s disease (HD), one with multiple myeloma (MM) and three with small cell lung Ó 2002 Blackwell Science Ltd

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Table I. Patients characteristics.

Case

Age

Sex

Diagnosis

Status at PBSCT

Conditioning regimen

MNC (108/kg)

CFU-GM (105/kg)

CD34 (106/kg)

Neutrophil recovery

Platelet recovery

1 2 3 4 5 6

47 47 18 40 34 44

M F M M M M

ALL NHL NHL AML AML NHL

CR1 CR1 CR1 CR1 CR2 CR1

3Æ7 2Æ6 3Æ1 2Æ6 2Æ3 2Æ0

1Æ6 14Æ9 5Æ6 3Æ0 4Æ6 2Æ6

3Æ6 19Æ4 9Æ7 3Æ6 2Æ5 2Æ6

17 10 10 11 14 11

23 15 12 63 40 100

7 8 9 10 11 12 13 14 15 16 17 18 19* 20 21* 22*

51 37 39 49 53 32 49 38 20 34 42 19 51 65 59 45

F M M F M M F F M M F F M M F M

MM NHL NHL NHL NHL AML NHL NHL NHL AML AML HD SCLC AML NHL NHL

CR1 CR1 CR1 CR2 CR1 CR1 CR3 PR PR CR1 CR1 CR1 CR1 CR1 PR PR

7Æ3 1Æ5 2Æ7 2Æ8 3Æ4 2Æ5 4Æ2 1Æ4 2Æ2 2Æ2 3Æ2 3Æ1 3Æ8 1Æ9 2Æ5 2Æ6

2Æ8 5Æ2 13Æ5 8Æ5 6Æ1 5Æ1 6Æ5 5Æ2 16 4Æ1 4Æ7 6Æ7 2Æ5 1Æ0 1Æ0 0Æ6

1Æ6 13Æ6 15Æ5 28Æ5 8Æ1 10Æ7 6Æ5 31Æ1 10Æ9 1Æ6 3Æ2 2Æ8 3Æ8 1Æ2 2Æ5 2Æ6

10 11 12 12 13 14 11 17 9 13 11 14 8 12 10 10

37 18 9 8 14 15 20 13 15 105 21 11 17 102 15 17

23* 24

56 53

M F

SCLC NHL

CR1 CR1

2Æ3 1Æ6

1Æ0 9Æ4

2Æ3 8Æ7

11 9

14 14

25

49

F

SCLC

CR1

CY/Ara-C/TBI CY/MCNU/ETP CY/MCNU/ETP BU/CY BU/ETP CY/MCNU/ETP/ CBDCA MCNU/ETP/MEL CY/MCNU/ETP CY/MCNU/ETP CY/MCNU/ETP CY/MCNU/ETP BU/CY CY/MCNU/ETP CY/MCNU/ETP ETP/TESPA BU/CY BU/CY CY/MCNU/ETP IFO/ETP/CBDCA BU/CY BU/CY/CA CY/MCNU/ETP/ CBDCA IFO/ETP/CBDCA CY/MCNU/ETP/ CBDCA IFO/ETP/CBDCA

3Æ7

7Æ0

9Æ4

10

17

*Purified CD34+ PBSCT. MNC, mononuclear cells infused; CFU-GM, granulocyte macrophage-colony forming units infused; CD34, CD34+cells infused; F, female; M, male; ALL, acute lymphoblastic leukaemia; NHL, Non-Hodgkin’s lymphoma; AML, acute myelogenous leukaemia; HD, Hodgkin’s disease; SCLC, small cell lung cancer; MM, multiple myeloma; CY, cyclophosphamide; Ara-C, cytarabine; ETP, etoposide; MCNU, ranimustine; BU, busulphan; CDBCA, carboplatin; MEL, melphalan; IFO, ifosphamide; TBI, total body irradiation; TESPA, thiotepa; CR, complete remission; PR, partial response; neutrophil recovery, day to reach more than 0Æ5 · 109/l; platelet recovery, day to reach more than 5 · 109/l.

cancer (SCLC). Patient characteristics are shown in Table I. Before the start of the study, informed consent was obtained from all patients. Mobilization and procurement of PBSCs. Haematopoietic stem cells were mobilized into peripheral blood by high-dose chemotherapy consisting of cytosine arabinoside (Ara-C) 12 mg/m2, cyclophosphamide (CY) (4 mg/m2), or etoposide (VP16) 1Æ5 or 2 g/m2 followed by 50 lg/m2 of recombinant human granulocyte colony-stimulating factor (rhG-CSF) (Shimazaki et al, 1992; Ashihara et al, 2000). RhG-CSF was subcutaneously administered, beginning on the second day after the last course of chemotherapy, and continued until the completion of leucapheresis. Peripheral blood mononuclear cells were collected on 2–3 consecutive days during the recovery phase from myelosupression using a CS3000 blood cell separator (Fenwal, Deerfield, IL). Leucapheresis was usually started when the number of CD34+ cells reached 20/ll in peripheral blood, as determined by flow cytometry. The leucapheresis product was stored with 5% dimethylsulphoxide (DMSO), 6% hydroxyethyl starch (HES)

and 4% human albumin. Cells were transferred into 100-ml freezing bags and cryopreserved at )130°C. In four patients, two with NHL and two with SCLC (cases 19, 21, 22 and 23), CD34+ cells were separated from the total PBSC using an Isolex system (Baxter Healthcare Immunotherapy Division, Round Lake, IL) and stored. Flow cytometric analysis. Cells were washed twice with phosphate-buffered saline (PBS) containing 2% fetal calf serum (FCS; Flow Laboratories, McLean, VA) and 0Æ1% sodium azide (PBS-azide), then incubated with 750 lg of purified immunoglobulin G (IgG; Green Cross, Tokyo, Japan) for 30 min at 4°C to block Fc receptors before the addition of specific antibody. Approximately 106 cells were resuspended in 1 ml of PBS-azide, and then incubated with monoclonal anti-CD34-phycoerythrin (PE) (Becton Dickinson, San Jose, CA) and CD90-fluorescein isothiocyanate (FITC) (PharMingen, San Diego, CA). In total, 13 patients (cases 1–13) were analysed for the expression of stem and immature progenitor markers as follows: anti-CD34-FITC and AC133-PE (Becton Dickinson), anti-CD34-PE and CD38-FITC (Becton

Ó 2002 Blackwell Science Ltd, British Journal of Haematology 117: 238–244

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Dickinson), and anti-CD34-PE and HLA-DR-FITC (Becton Dickinson) for two-colour analysis. As negative controls, IgG1-PE and IgG1-FITC (Becton Dickinson) were used. Cells were washed twice and resuspended in 0Æ5 ml of PBS-azide. Flow cytometric analysis was performed using an EPICS Profile flow cytometer (Coulter, Hialeah, FL) as described previously (Inaba et al, 1994). Statistical analysis. The Spearman rank correlation was used to analyse the data. A value of P < 0Æ05 was accepted as statistically significant. RESULTS Early haematopoietic recovery after PBSCT Following myeloablative pre-transplant conditioning, a mean of 5Æ1 · 105 CFU-GM/kg (range 0Æ6–16Æ0) and 3Æ8 · 106 CD34+ cells/kg (range 1Æ6–31Æ1) were infused into patients (Table II). The median numbers of days to reach neutrophils > 0Æ5 · 109/l and platelets > 50 · 109/l were 11 d (range 8–17) and 15 d (range 8–105) respectively. Six patients (cases 4–7, 16 and 20) showed a delayed recovery of platelets at more than 1 month after PBSCT. They received relatively low numbers of CD34+ cells (Table I). The number of CD34+ cells and their stem and immature progenitor cell subsets infused are shown in Table II. The numbers of CD34+/AC133+, CD34+/CD38– and CD34+/HLA-DR– cells were analysed in 13 patients. A large variation was observed in the number of CD34+/ CD90+ cells and CD34+/AC133+ cells in each case. Late haematopoietic reconstitution after PBSCT The median values of haemoglobin (Hb), white blood cell (WBC) and platelet counts in peripheral blood at 3, 6, 9 and

Table II. Number of cells infused.

Median n ¼ 25 CFU-GM (· 105/kg) CD34+ cells (· 106/kg) CD34+/CD90+ cells (· 104/kg) n ¼ 13 CD34+/AC133+ cells (· 104/kg) CD34+/CD38– cells (· 104/kg) CD34+/HLA-DR– cells (· 104/kg)

Range

12 months after PBSCT are shown in Table III. The number of samples analysed between 6 and 12 months were reduced because some patients received tandem transplants during the first 6 months after PBSCT, or relapsed and the blood counts could not be evaluated. WBC counts were constant after 3 months whereas Hb and platelet counts increased and became stable 6 months after PBSCT. Relationship between the number of stem and immature progenitor cells infused and late haematopoietic reconstitution after PBSCT The relationship between the numbers of infused CD34+ cells, and their subsets expressing CD90, AC133, or not expressing CD38 and HLA-DR, and the Hb, WBC and platelet counts at each month after PBSCT were analysed. Significant positive correlations are summarized in Table IV. CD34+ cells and their subsets CD34+/CD90+ and CD34+/ AC133+ cells infused correlated to platelet counts at 3 months after PBSCT (Table IV, Fig 1). Among them, the CD34+/CD90+ cell dose was most strongly correlated to platelet counts (r ¼ 0Æ756, P < 0Æ0001). No correlation was observed between another CD34+ subset (CD34+/ CD38– and CD34+/HLA-DR– cells) and haematopoietic reconstitution at 3 months after PBSCT (data not shown). The number of CD34+/CD90+ cells were also correlated to platelet counts at 6, 9 and 12 months (Table IV, Fig 1). In addition, CD34+/CD90+ cells correlated to Hb level at 12 months after PBSCT (Table IV). CD34+/AC133+ cells correlated to the WBC count at 3 months and CD34+/ CD38– cells correlated to the WBC count at 9 months after PBSCT (Table IV). With regard to the threshold dose for long-term engraftment, eight out of 10 (80%), three out of five (60%), two out of three (66%) and two out of two (100%) patients who received less than 80 · 104/kg of CD34+/CD90+ cells did not reach platelet counts of 100 · 109/l at 3, 6, 9 and 12 months after PBSCT respectively (Fig 1). DISCUSSION

5Æ1 3Æ8 107Æ7

0Æ6–16Æ0 1Æ2–31Æ1 9Æ6–476Æ0

258Æ0 26Æ3 63Æ1

19Æ2–1291Æ0 3Æ1–129Æ0 21Æ2–155Æ0

In the present study, we demonstrated that the number of immature haematopoietic stem cells infused expressing CD34+/CD90+ best predicted platelet reconstitution during the first 12 months after PBSCT, although the total number of CD34+ cells and those co-expressing AC133 were also correlated. Whereas the CD34+/AC133+ cell dose correlated with the WBC count at 3 months after PBSCT, no other CD34+ cells nor their subsets correlated with WBC counts or

Table III. Haematopoietic reconstitution after PBSCT.

WBC (· 109/l) Hb(g/dl) PLT (· 109/L)

3 months

6 months

9 months

12 months

4Æ6 (3Æ5–10Æ1) 11Æ3 (6Æ8–14Æ4) 150 (20–399) (n ¼ 25)

5Æ4 (3Æ5–11Æ8) 12Æ1 (7Æ1–16Æ1) 197 (24–306) (n ¼ 14)

4Æ9 (3Æ7–8Æ6) 13Æ0 (9Æ3–15Æ7) 191 (35–308) (n ¼ 11)

5Æ1 (4Æ6–6Æ8) 12Æ8 (8Æ8–15Æ2) 208 (44–349) (n ¼ 9)


CD34 Subset and Late Haematopoietic Reconstitution after PBSCT

241

Table IV. Correlation of subsets of cells with WBC, Hb and platelet counts after PBSCT.

3 months

Cells/kg WBC CD34+/AC133+ CD34+/CD38– Hb CD34+/CD90+ Plt All CD34+ CD34+/CD90+ CD34+/AC133+

Correlation coefficient

6 months

P value

9 months

12 months


P value


P value


P value

0Æ591 0Æ110

0Æ0317 0Æ7278

0Æ539 0Æ759

0Æ2966 0Æ0850

0Æ547 0Æ843

0Æ2870 0Æ0331

0Æ480 )0Æ466

0Æ4598 0Æ4755

0Æ029

0Æ8921

0Æ268

0Æ3631

0Æ375

0Æ2645

0Æ732

0Æ0222

0Æ598 0Æ756 0Æ703

0Æ0012 £ 0 0001 0Æ0057

0Æ495 0Æ636 0Æ778

0Æ0717 0Æ0127 0Æ0719

0Æ635 0Æ664 0Æ908

0Æ0339 0Æ0238 0Æ0086

0Æ565 0Æ850 0Æ763

0Æ1171 0Æ0021 0Æ1558

Æ

Figures in bold signify statistical significance.

Fig 1. Correlation between the number of CD34+/CD90+ cells infused and platelet count after PBSCT. M, months.

Hb levels at this time. In addition, the CD34+/CD90+ cells correlated with Hb level at 12 months after PBSCT, and the CD34+/CD38– cells correlated to the WBC count at 9 months after PBSCT. Few studies have been reported on the effect of stem and immature progenitor cell doses on the long-term haematopoietic recovery after PBSCT (Haas et al, 1995b; Kiss et al, 1997; Amigo et al, 1999; Perez-Simon et al, 1999; Duggan et al, 2000). Kiss et al (1997) reported that infused CD34+ cells correlated with the Hb levels at 180 and 360 d, and platelet count at 180 d after PBSCT. Amigo et al (1999) also reported that the CD34+ cell dose correlated with both 6 and

12 month Hb and platelet count but not with WBC count. In addition, Duggan et al (2000) also reported a correlation between the CD34+ cell dose and the long-term engraftment of WBCs and platelets. However, we found a correlation between the CD34+ cell dose and the platelet count during the first 9 months after PBSCT (weak correlation at 6 months; P ¼ 0Æ0717). This observation, together with others, suggested that platelets are the most affected blood parameter after PBSCT and thrombopoiesis is the best indicator of graft function (Tricot et al, 1995). Committed progenitor cells, expressing myeloid (CD33, CD13), erythroid (CD71) and megakaryocytic (CD41,


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CD61) markers have shown some correlation with early engraftment after PBSCT (Dercksen et al, 1995; Feng et al, 1998; Stewart et al, 1999). However, late haematopoietic reconstitution is considered to depend on the dose of stem and immature progenitor cells. Hirao et al (1994) reported that the number of infused long-term culture colonyforming cells (LTC-CFC)/kg did not correlate with early recovery of granulocytes, but significantly correlated with the recovery of granulocytes after a transient decrease. Therefore, we examined the number of stem and immature progenitor cells using two markers, CD90 and AC133, which identify immature subsets of CD34+ cells. CD90 (Thy-1), a 25 to 35-kDa molecule, is expressed by 1–4% of human fetal liver cells, cord blood cells and bone marrow cells (Craig et al, 1993; Mayani & Lansdorp, 1994). CD34+/CD90+ cells have a capacity to reconstitute human haematopoiesis in severe combined immunodeficient (SCID) mice, and initiate long-term haematopoiesis in vitro (Baum et al, 1992; Craig et al, 1993). Owing to the discrepancy in the number of CD34+/CD90+ cells relative to CD34+/CD38– cells, these cells are considered to contain more than just primitive progenitor cells (Humeau et al, 1996). CD34+/ CD90+ cells are mobilized into blood at a maximum level a few days earlier than peak mobilization of the total CD34+ cells (Haas et al, 1995b; Murray et al, 1995; Stewart et al, 1995). Another marker is AC133, a novel 5-transmembrane cell surface antigen, which presents on haematopoietic stem and progenitor cells (Miraglia et al, 1997; Yin et al, 1997). AC133 is expressed on 20–60% of CD34+ cells but not on mature blood cells. CD34+/AC133+ cells contain the majority of the CFU-GM, a proportion of the CFU-Mix, a minor population of BFU-E and the long-term repopulating stem cells (Yin et al, 1997). In the present study, we demonstrated that infused CD34+/CD90+ cells most strongly correlated with platelet counts during 12 months and Hb level at 12 months after PBSCT but not with WBC. Perey et al (1998) and Stewart et al (2000) demonstrated that the CD34+/CD90+ cell dose does not predict early engraftment, but they did not examine its effect on the late haematopoietic recovery. Only one study has demonstrated the effect of the CD34+/CD90+ cell dose on the late engraftment after PBSCT. Duggan et al (2000) reported the correlation of the CD34+/CD90+ cell dose with long-term (median 6 months) engraftment of WBC (P ¼ 0Æ009) and platelets (P ¼ 0Æ0011). However, they showed that the total CD34+ cell dose infused was superior to the subsets of CD34+ cells including CD34+/ CD90+ cells in predicting 6 month blood counts following PBSCT. In the present study, the total CD34+ cell dose was inferior to CD34+/CD90+ cells. This difference may be partly related to the small number of patients studied or the dose of the CD34+/CD90+ cells infused. The median numbers of the CD34+/CD90+ cell dose in the present and previous studies were 1Æ08 · 106/kg and 3Æ09 · 106/kg respectively. The peak of CD34+/CD90+ cells appeared in the peripheral blood a few days earlier than that of CD34+ cells, so the percentage of CD34+/CD90+ cells in the apheresis product was different on the day of apheresis in each study (Murray et al, 1995; Stewart et al, 1995). With regard to the

threshold value of the CD34+/CD90+ cell dose for durable engraftment, more than 60% of patients who received less than 80 · 104/kg of CD34+/CD90+ cells did not reach a platelet count of 100 · 109/l at 3, 6, 9 and 12 months after PBSCT in the present study (Fig 1). These observations are compatible with those of Michallet et al (2000) who demonstrated that 80 · 104/kg of CD34+/Lin–/CD90+ cells was a threshold dose for fast and durable haematopoietic engraftment. While the CD34+/AC133+ cells infused also correlated with platelet counts during the first 9 months after PBSCT (weak correlation at 6 months; P ¼ 0Æ0719) in the present study, CD34+/AC133+ cells infused also correlated with WBC at 3 months. No studies have reported on the correlation between the CD34+/AC133+ cell dose and the long-term engraftment. Further studies are required on the effect of CD34+/AC133+ cells on long-term engraftment. Other primitive progenitor cells, such as CD34+/CD38– cells and CD34+/HLA-DR– cells, did not show any correlations with peripheral blood cell counts after PBSCT, except CD34+/CD38– cells with WBC count at 9 months. With regard to the CD34+/CD38– cell dose, Duggan et al (2000) reported a correlation with platelets several months after PBSCT. This difference might be as a result of the small number of samples in the present study. The number of CD34+/CD33– cells, another primitive progenitor cell subset, was reported to provide information about the quality of the graft necessary for long-term platelet engraftment (Millar et al, 1998), but unfortunately we did not examine this subset. In conclusion, as only 13 patients had stem cell markers other than CD90 studied, the results of this study do not entirely eliminate the possibility of other subsets being equally predictive for late haematopoietic recovery. However, CD34+/CD90+ cells in PBSCs were a better predictor of platelet recovery than the total dose of CD34+cells, and 80 · 104/kg of CD34+/CD90+ cells is the minimum required dose for durable long-term engraftment. Therefore, the measurement of CD90+ cells in CD34+ cells is useful for the evaluation of the quality of grafts in PBSCT. REFERENCES Amigo, M.L., del Canizo, M.C., Caballero, M.D., Vazquerz, L., Corral, M., Vidriales, B., Brufau, A. & San Miguel, J.F. (1999) Factors that influence long-term hematopoietic function following autologous stem cell transplantation. Bone Marrow Transplantation, 24, 289–293. Ashihara, E., Shimazaki, C., Okano, A., Hatsuse, M., Inaba, T. & Nakagawa, M. (2000) Feasibility and efficacy of high-dose etoposide followed by low-dose granulocyte colony-stimulating factor as a mobilization regimen in patients with non-Hodgkin’s lymphoma. Haematologica, 85, 1112–1114. Baum, C.M., Weissman, I.L., Tsukamoto, A., Buckle, A. & Peault, B. (1992) Isolation of a candidate human hematopoietic stem-cell population. Proceedings of National Academy of Science of the United States of America, 89, 2804–2808. Bensinger, W.I., Longin, K., Appelbaum, F., Rowley, S., Weaver, C., Lilleby, K., Gooley, T., Lynch, M., Higano, T., Klarnet, J., Chauncey, T., Storb, R. & Buckner, C.D. (1994) Peripheral blood


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