RESULTADOS DE LOS ENSAYOS DE CARGA EN MUROS

Fifty-eight patients with relapsed or resistant lymphoma and 11 myeloma patients were studied. The patient demographics are summarised in Table 8.1.

The ablative therapy used for the lymphoma patients was the BEAM protocol (Chopra et al., 1993; Linch et al., 1993) ( BCNU 300mg/m^/V. on day 1, etoposide 200mg/mVday iv days 2 - 5, (total dose 800mg/m^ ) cytosine arabinoside 200mg/m" b.i.d on days 2 - 5 (total dose 1600mg/m") and melphalan 140mg/m" iv. on day 6.

The ablative therapy for the myeloma patients was 100 to 140mg /m^ melphalan and 8 to 9 Gy of fractionated total body irradiation (Jagannath et al., 1992).

Table 8.1 Patients receiving unfractionated or CD34+ ceii selected peripheral blood stem cells.

Clinical details Whole PBSC

(n= 59) Purified CD34+ cells (n=10) Hodgkin’s disease 42 2 NHL 10 3 Myeioma 7 5 Maies : Females 41 : 18 4 : 6 Median Age (range)

Previous wide field irradiation

31 (15-67) 44 (17-59)

Previous cycles of

5/59 0/10

chemotherapy median (range)

Bone marrow involvement at

7(3-17) 6(3-11)

time of PBSC harvest 0/59 0/10

8.2.2 PBSC mobilization and collection.

All patients were mobilized with low dose cyclophosphamide (1.5g/m^) given on the first day (“day 1”) followed by G-CSF given sca t lOpg/kg (filgrastim) or a single vial of lenograstim (263 pg/vial) 24hrs afterwards and daily thereafter until harvesting was complete. One to three apheresis collections were performed on days 8 to 12, on a COBE Spectra or Baxter CS3000, commencing when the recovery WBC first exceeded 5.0 xIO^L. The apheresis collection was then cryopreserved in an equal volume of autologous plasma containing 20% dimethyl sulphoxide (DMSG) followed by controlled rate freezing and storage in liquid nitrogen. When required the PBSC were thawed at the bedside at 40°C and returned via a fast running central catheter. In 10

patients, a single apheresis was subjected to CD34+ cell selection as described below. The CD34+ selection process resulted in a 90ml volume cell fraction which was concentrated and cryopreserved in two 4.5 mL aliquots as detailed in Chapter 7. When stem cell re-infusion was required, each individual vial was removed from its outer 15ml tube, thawed in a sealed bag in a water bath at 37°G and slowly reconstituted with warmed 8% human serum albumin in PBS up to a 30ml volume. The re-suspended cells were then drawn into a 50ml syringe and returned to the patient via a 3-way tap into a fast running intra-venous saline drip.

8.2.3 CD34+ cell selection

Single apheresis collections were processed for clinical scale CD34+ cell purification using a CEPRATE SC immunoaffinity column and associated microprocessor device using the manufacture’s protocol as detailed in Chapter 7. A 0.5ml aliquot of the purified CD34+ cell fraction was taken for cytocentrifuge preparations for blast cell morphology, CD34 numbers by alkaline phosphatase-anti-alkaline phosphatase (APAAP) immunoenzymatic staining and for flow cytometry and colony forming cell (CFC) cultures.

8.2.3 Progenitor evaluation

A sterile harvest sample was taken from the apheresis bag pilot line after repeated “stripping back” of the product into the bag which was continually mixed to ensure even cell suspension. This sample was then diluted 1/10 and 1/100 in RPMI containing 10% fetal calf serum and 20U/ml heparin. Direct harvest smears and these dilution’s were then used for a full blood count (STKR Coulter inc. Hialeah, FL.), manual white cell differential and progenitor measurement by CD34+ cell and CFC assays as detailed in the general methods materials Chapter 2 and for CD34+ cell selection specifically, in Chapter 7.

8.2.4 Accessory cell depletion. Smears from unfractionated harvest products, pre CD34+ cell processing harvest smears and corresponding cytospins from the purified CD34+ fractions were tightly wrapped in parafilm and stored at -20°C for batch staining. Monocytes were identified by esterase staining (Li et al., 1973) and T-cells by APAAP staining (Erber et a i, 1984) with a CD3 mouse monoclonal (UCH-T1, Dako Ltd., Bucks, England). Positively reacting cells were counted for each cell marker by examining an approximated 2000 cells with a “Miller’s squares” eyepiece counting graticule (Graticules Ltd., Tonbridge, Kent UK).

8.2.4 Cytokine assays. Tripotassium-EDTA specimens (Vacutainer, Becton Dickenson) were collected from patients before high dose therapy, (“pre”), on the day of stem cell infusion, (day 0) and on alternate days up to day 23. The samples were centrifuged at 3500g for 15 minutes within 4hrs of collection and plasma separated and stored at -20°C until required.

Plasma concentrations of all growth factors were evaluated by specific enzyme- linked immuno-adsorption assay (ELISA) kits (“Quantikine” , R+D systems- British Biotechnology, Cowly, Oxford, UK). The detection thresholds of the cytokines measured were; G-CSF 5.0 pg/ml, GM-CSF 20 pg/ml, IL-6 10 pg/ml, stem cell factor 5.0 pg/ml and MIP-1-alpha 3.0 pg/ml.

The detection method was similar for each assay. A mouse monoclonal antibody specific to the cytokine to be measured was provided adsorbed to 96 well polystyrene plates. Geometric dilution’s of plasma control recombinant human cytokine were added to the wells with test samples in duplicate, and incubated for 2 hours at room temperature. After washing the plate, a peroxidase conjugated goat -anti -mouse antibody was added for a further 2 hour incubation, followed by a second wash to remove un-reacting antibody. Tetramethylbenzidine was then added which is oxidised by peroxidase activity to a blue reaction product. The reaction was stopped after 20 minutes with 2N

sulphuric acid and absorbance read at 450nm vs. substrate blank on a microtitre plate reader (Dynatech MR700, Dynatech labs, ltd., Sussex, UK) . Absorbance of the test wells were compared with the standard curve and expressed as pg/ml. Means and SEMs were calculated for various time points for each of the two groups of patients (whole PBSC infused or CD34+ stem cells only) at pre, day 0, then grouped at intervals of days 1-3, 4-6, 7-9, 10-12,

13-15, 16-18 and finally days 19-21.

8.2.3 Statistical analysis of data.

Recovery times of patients given unfractionated PBSC were compared with those for patients who received purified CD34 cells by the Mann-Whitney U test for non-parametric, unpaired data on an IBM PC ( Number Cruncher Statistical System 5.x, Univ. Utah ).

8.3 RESULTS

8.3.1 CD34+ cell purification and accessory cell depletion

Peripheral blood progenitor cells from a single apheresis collection were purified on a CEPRATE SC immunoaffinity column and subsequently used as haematological support following high dose therapy in 10 patients. In this group, the median dose (range) of CD34+ cells infused was 3.0 xIOVkg (0.5- 6.9 x10®/kg) with a median purity of 81% (44-93%) (Table 8.2). The CD34 purification process resulted in a greater than 2 log depletion of monocytes and greater than 3 log depletion of T cells (Figure 8.1). Six patients were also studied who received whole PBSC from 2 or 3 apheresis collections. The comparative numbers of accessory cells infused in this group are also shown in Figure 8.1 with the accessory cells present in individual harvests compared with that of the pooled harvests re-infused in this group.

Table 8.2 Processing results of CD34+ cell selection: Patient number Diag H arvest MNC x 1 (f values CD34% CD34 purity

Post processing results

CD34 CD34 x K f/k g yield CFC yield 1 HD 23 1.4% 81% 3.5 60% 43% 2 HD 42 1.8% 88% 6.1 48% 80% 3* NHL 23 2.8% 54% 1.9 21% 34% 4* NHL 13 2.4% 87% 1.6 30% 12% 5* NHL 19 1.5% 80% 2.8 102% 84% 6* MM 22 1.1% 77% 3.1 78% 53% T MM 19 1.6% 86% 2.8 75% 33% 8* MM 50 2.4% 93% 6.9 50% 53% 9 MM 19 1.3% 44% 0.5 11% 10% 1 0 MM 31 1.6% 61% 3.1 47% 45% M e d ia n s 2 3 1 .6 % 8 1 % 3 .0 4 9 % 4 4 % (range) (13-50) (1.1-28) (44-93) (0.5-6.9) (11-102) (10-84) Go o L ' ^ (o \2_ < 3 2 - U I V 6.C

Monocyte numbers Whole PBSC CD34 selected T-cells numbers whole PBSC CD34 selected c =3 O Ü 0 Ü Individual Total harvests returned Pre-Post CD34 selection Individual Total harvests returned Pre-Post CD34 selection

Figure 8.1 Accessory cell depletion following CD34+ cell purification. Results are shown for a single and total harvest infused for the whole PBSC group. This is compared with the single collection used for the CD34 purified patients.

8.3.2 Cytokine profiles following stem cell Infusions.

Serial plasma G-CSF levels were measured in 12 patients following haemopoietic stem cell infusions and IL-6 in 11. The G-CSF and IL-6 profiles are almost a mirror image of the neutrophil and platelet levels, both cytokines rising as the blood counts fall and falling to baseline levels as the blood counts recover (Figure 8.2). With both cytokines there was an abrupt rise in plasma levels between days 5 and 14 post stem cell infusion (Figures 8.3A and 8.3B). Six patients had received purified CD34+ cells and 6 received whole PBSC and there was no significant difference between these two groups. The cytokine levels were not different between those patients with lymphoma receiving BEAM chemotherapy and those with myeloma receiving melphalan/TBI conditioning (data not shown). The median number of cycles of chemotherapy previously given in the 6 patients receiving purified CD34+ cells was 5 compared to 8 in the recipients of whole PBSC and one of the latter group had received wide field irradiation compared to none in the former group.

Serial plasma stem cell factor and GM-CSF levels were also measured in 11 patients. There was no detectable change in these levels at all time points (data not shown). Similarly, plasma MIP1-alpha levels were measured in five patients and showed no significant changes over the recovery period (data not shown).

Neutrophil count X109/L--- r 1000 4 - 100 # v preO 2 4 6 8 10 12 14 16 18 20 22 Platelets xIO /L G-CSF (pg/ml) IL-6 (pg/ml)

Days post stem cell infusion

Fig 8.2 Serial cytokine changes associated with haematological recovery post high dose therapy and stem cell infusion. Median values shown, n=12

- o - ’ " purified CD34+ cells 10000-1 _{whole PBSC} E D) Q. 1000- u_ CO Ü Ô CO E cn jO CL 1 00- 12 0 3 6 9 15 18 Pre

Days post stem cell infusion

E Q. CO CD > _0) C£) CO E CO _C0 CL 100-1 1 / 9 12 15 18 21 24 Pre 0 3 6

Days post stem cell infusion

Figure 8.2. Endogenous plasma cytokine levels post PBSCT. A) G-CSF and B) IL-6

8.3.4 Haematological recovery following infusion of purified cells.

Ten patients were rescued from high dose chemo/radiotherapy by purified CD34+ cells from a single apheresis. This group contained the 6 patients on whom the cytokine profile was measured and a further 4 patients. Five patients had lymphoma (2 Hodgkin's disease and 3 non-Hodgkin's lymphoma) and 5 had multiple myeloma

Although none of these patients was given G-CSF immediately following stem cell re-infusion, two patients, patient numbers 1 and 6 were given a single injection of G-CSF on days 18 and 22 respectively because of delayed neutrophil recovery. Although neutrophils >0.1 x10^/L were detectable by day 11 the subsequent rise was slow. In both patients the neutrophil count was >0.5 xIO^/L the following day after the G-CSF injection and no further G-CSF was given. The neutrophil count subsequently remained above 0.5 xIO^/L.

The recovery data is shown in Table 8.3 and a more detailed comparison with the recovery in 59 patients (39 with Hodgkin’s disease, 14 with non-Hodgkin’s lymphoma and 6 with myeloma) following whole PBSCT is shown in Figures 8.4A and 8.4B in which recovery is plotted against the total GM-CFC dose infused. There was no discernible difference in recovery between those patients with lymphoma or multiple myeloma. None of the recipients of whole PBSCT received G-CSF immediately after stem cell infusion, although it was given to 5 patients beyond day 11 because of slow neutrophil recovery. Overall the neutrophil recovery to 0.5 x10®/L was marginally slower in the recipients of purified CD34+ cells than in recipients of whole PBSC ( p<0.05 ). Recovery is however related to dose of GM-CFC infused, with a higher proportion of slow recoveries in recipients of < 3.5 xIOVkg. This lower dose was more frequent in the recipients of purified CD34+ cells as they were derived from a single apheresis whereas the whole PBSC recipients received an average of 2 collections. In addition, CD34 purification results in some

losses (approximately 50%). If only those patients who receive a GM-CFC dose of < 3.5 x1 OVkg are considered, there is no significant delay associated with the use of purified CD34 cells. Platelet recovery was not significantly different between the recipients of whole PBSC and purified CD34 cells.

Table 8.3 Haematological recovery of patients receiving CD34+ purified cells compared to patients receiving whole PBSC

Patient number diag Days of ANC <0.1x10^/L Days to ANC 0.5x10^/L Days to Platelet indep. Platelet units given Days to Platelets >50x10®/L 1 * HD 6 23 15 10 19 2 HD 4 12 13 10 17 3 NHL 2 13 9 10 11 4 NHL 5 17 21 55 22 5 NHL 9 18 14 30 21 6 * MM 11 19 41 105 44 7 MM 5 18 13 20 20 8 MM 6 12 11 10 12 9 MM 7 16 18 25 41 1 0 MM 7 16 8 35 13 CD 3 4 6 1 6 . 5 1 3 . 5 2 2 . 5 2 0 med/range (4-9) (11 -23) (9-41) (11 -41) (10-105) Whol e 6 1 2 1 1 1 5 2 2 PBPC: (n =59) (1-11) (6-27) (6 - >85) (5 - 230) (9->100)

*A single injection of G-CSF was administered to patient 1 on day 22 and a single dose to patient 6 on day 18

O) o X m d A O z < o (/) S' Q 100 -1 purified CD34+ cells: n=10 whole PBSC: n=59 8 0 - 6 0 “ 4 0 - 0 5 10 15 20 25

B

Q) O C 0) "O c 0) Q. 0) "D C CL O (/) Q 100-1 <X> 80- 4 0 - 2 0- o o 0 5 10 15 20 25 GM-CFC dose x 105/kg

Figure 8.4 Haematological recovery of A) neutrophils and B) platelets in patients receiving whole or CD34+ cell purified PBSCT

8.4 DISCUSSION

This study confirms that following high dose chemotherapy or radiotherapy followed by PBSCT there is a marked rise in plasma G-CSF and IL-6 levels. The levels of G-CSF peaked around day 5 at approximately 1 ng/ml and IL-6 of approximately 50pg/ml. The peak G-CSF levels are in accord with data published by Baiocchi et aland Ho e ta ! (Baiocchi eta!., 1993; Ho at a!., 1994), but are somewhat lower than that reported by other groups (Haas et a!., 1993; Kawano eta!., 1993; Testa et a!., 1994) This may represent methodological differences but is also likely to represent the different patient groups that have been studied. Testa et al, found that the levels achieved were related to the underlying disease (and thus the pre-transplant treatment given) and that patients with ovarian cancer gave higher levels than those with haematological malignancies (Testa et al., 1994). In accord with this, we have studied one patient receiving high dose therapy for breast cancer and found peak G-CSF levels on days 4 to 8 of approximately 5.0 ng/ml.

The levels of stem cell factor did not change significantly in the post-transplant period and this is in accord with the report from Testa (Testa et al., 1994). The levels of GM-CSF were below the levels of detection following transplantation, and similar results have been reported by others (Ho et al., 1994; Kawano et al., 1993). Testa, in contrast, did find a post transplant rise in GM-CSF in a proportion of patients (Testa et al., 1994), The reason for this discrepancy is not clear.

In this study, we observed no significant difference in the level of G-CSF of IL-6 achieved between patients with lymphoma or myeloma although the conditioning regimen used for the two diseases was different. There was also no difference in the cytokine profiles of the recipients of purified CD34+ cells compared to those of whole PBSC even though the former received approximately 3 logs less monocytes and even fewer T-cells. This strongly

suggests that such re-infused accessory cells do not contribute to the production of cytokines during the neutropaenic and thrombocytopaenic period.

The recipients of accessory depleted progenitor cells had marginally slower neutrophil recovery than the whole PBSC recipients, but this could be largely attributed to the smaller numbers of progenitors (GM-CFC) that were re-infused to these patients illustrating that there is a threshold number of progenitors required below which the risks of slow engraftment increase (Bensinger et al.,

1995; Schwartzberg et a!., 1993; Tricot et a!., 1995; Weaver et a!., 1995; Weaver et a!., 1997). One of the recipients of the purified CD34+ cells had delayed platelet recovery, which is in accord with the experience obtained with whole PBSC re-infusions. These data indicate that re-infused accessory cells do not make a major contribution to the rapid haematological recovery seen with PBSCT compared to ABMT although as this was not a randomized study a minor contribution of accessory cells cannot be fully excluded. It must also be noted that of the 10 patients who received purified PBSC, 5 were conditioned with BEAM chemotherapy which is not fully myeloablative. Some caution must therefore be exercised in determining any accessory cell requirements for the engraftment/proliferation of the most primitive stem cells.

As the more rapid regeneration is not apparently due to accessory cells in the PBSC, this implies it is due to the infusion of a functionally different subset of progenitor cells.

None of the patients in this study received growth factors immediately following stem cell re-infusion, and it seems highly likely from the recovery data presented in the previous chapters and recent randomized trials (Klumpp et a!.,

1995; Linch et a/., 1997) that the recovery of neutrophils may have been accelerated by the administration of G-CSF.

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