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6 . due to atrial flutter. PD= progressive disease, NA=not assessable, SD=stable disease.

Following the second course, responses were not observed and therapy with rlL-2 was discontinued. Patients entered into the single five day course of rlL- 2 were not considered eligible for response.

8:4. Biological Evaluation: Leucocyte counts.

8:4:1. Pre-treatment lymphocyte counts.

Differential blood counts were performed before and during treatment with rlL- 2. Patients with metastatic breast cancer treated on protocol EC-L2041 had a low pre-treatment lymphocyte count compared with patients on the malignant melanoma/renal cell carcinoma protocols (medians= 0.5 and 0.9 respectively p<0.03 Mann-Whitney). The median pre-treatment lymphocyte count in patients with breast cancer who had not received chemotherapy for metastatic disease was I.SxIO^/L. Patient numbers in the two groups of patients with breast cancer were too small for a valid comparison to be made.

8:4:2. Lymphocyte counts during rlL-2.

Lymphocyte counts during infusion of rlL-2 in the different patient groups are illustrated in figure 8:4:1 (overleaf). Twenty four hours following the start of continuous infusion rlL-2, the peripheral lymphocyte count fell in all the patient groups studied and remained below pre-treatment levels for the duration of the infusion. Before the start of subsequent infusions (48 to 72 hours following cessation of rlL-2) lymphocyte levels were higher compared with values taken on the final day of the rlL-2 infusion in all groups studied. In patients on the escalating dose schedule this lymphocyte rebound did not vary greatly even at the highest projected dose, being a median of 1.7, 1.25, 1.6, and I.IxlO ^/L following the first, second, third and fourth infusions of rlL-2 respectively. The rebound lymphocytosis was higher in the other groups being median of 2.68, 5.34 and 2.15x10^/1 in patients treated on the melanoma, renal cell carcinoma and breast cancer protocols respectively. Statistical comparisons between groups are inappropriate because of small patient numbers. It was

not possible to assess the rebound lymphocytosis in patients receiving two five-day infusions owing to discharge home of all but two patients.

Figure 8:4:1. Changes in iymphocyte counts during infusions of rlL-2.

metastatic breast cancer: ECL2041 2-

0 7 14 21 2 8

8

metastatic renal cell carcinoma: ECMP001 6 4 2 0 0 7 14 21 28 Ü

I

S I Q. E 6- 4 - 2- 0 7 14 21 2 8 4

metastatic breast cancer: C74. 2

0

0 7 14 21 28

8:4:3. Eosinophil counts during rlL-2.

Changes in eosinophil counts during rlL-2 are illustrated in figure 8:4:2

Figure 8:4:2. Changes In eosinophil counts during infusions of rlL-2.

metastatic breast cancer: C74 ./*voo~oO~ I 1 I ■ 1■ ^ . i i i . ^ ... ^ ,

14 21

Time (days)

28

metastatic breast cancer: ECL2041 8-

6-

4 -

2-

0 7 14 21 2 8

metastatic renal cell carcinoma: ECMP001 4 - O) o 2- X c₃ O Ü . c CL O c s LU 0 7 14 21 2 8

metastatic melanoma: ECMP003 4 -

2-

Pre-treatment eosinophil counts were low and changed little during the first infusion period in all protocols. During the second infusion period eosinophil counts increased considerably and were similar in all groups. In patients on the escalating dose schedule, eosinophil levels during the second infusion period were similar to those observed in the other patient groups even though the projected dose of rlL-2 was lower. The highest levels of eosinophils were noted during the third infusion period and not at the final dose level.

8:5. Biological Evaluation: Cytokine induction.

8:5:1. TNF-a and TNF soluble receptors.

TNF-a may be important in modulating the anti-tumour effect of rlL-2 but may also mediate some of the toxicities of rlL-2. Proteins formed by proteolytic cleavage of the extracellular domain of the low (p55) and high (p75) molecular weight TNF receptors are capable of binding and inhibiting the biological effects of TNF-a. Since increases in these binding proteins have been demonstrated during treatment with TNF-a, and therapy with rlL-2 is known to induce TNF-a, levels of TNF-a and its soluble receptors were measured during treatment with rlL-2.

TNF-a levels were measured by a commercially available IRMA kit in 22 patients receiving rlL-2 by infusion as part of the protocols described previously. Soluble receptors sTNF-Rp55 and sTNF-Rp75 were also measured by ELISA in a sub-group of 13 patients during therapy.

The mean pre-treatment level of TNF-a in this group of patients was not significantly different from level found in 25 normal controls (8.3+1.5 pg/ml vs

7.4+0.6 pg/ml), table 8:5:1. The mean pre-treatment level of sTNF-Rp55 was however significantly higher compared with 53 normal controls (1.6±0.1 ng/ml

vs 0.7 ng/ml, p<0.001). Similarly, the mean pre-treatment level of sTNF-Rp75 in patients was 3.1 ±1.4 ng/ml compared with 2.1+ pg/ml in normal controls (p<0.001).

Table 8:5:1. Circulating TNF-a and TNF binding proteins in normals and patients with advanced cancer before rlL-2.

Group Normals TNF (pg/ml±s.e.) 7.4±0.6 (n=25) Pre-treatment (n=22) 8.3±1.5 sTNF-Rp55 (ng/ml±s.e.) 0.7±0.1 (n=53) 1.6±0.2* sTNF-Rp75 (ng/ml±s.e.) 2.110.1 (n=53) 3.110.3'

statistically significantly different from normai samples (p<0.001, t-test).

The induction of TNF-a was assessed in all patients receiving rlL-2 and TNF levels by patient group are illustrated in figure 8:5:1. In all cases, treatment with rlL-2 led to induction of TNF. Induced levels of TNF rose throughout the period of rlL-2 infusion but levels fell rapidly (assessed 48 to 72 hours) following discontinuation of rlL-2. TNF levels in patients treated on the escalating dose schedule were low compared with the other groups even at the highest projected level of 18x 1 0^ I.U ./m ^ . Indeed the highest levels of TNF-a in this group were recorded during the intermediate dose level 15x10®

I.U ./m 2 and in two patients peak TNF levels fell with increasing doses of rlL-2.