Safety, tolerability, pharmacokinetics and pharmacodynamics of AZD8055 in advanced solid tumours and lymphoma.

BACKGROUND: This study assessed the safety, tolerability, pharmacokinetics and pharmacodynamics of the first-in-class dual mammalian target of rapamycin complex (mTORC)1/mTORC2 inhibitor, AZD8055. METHODS: Patients with advanced solid malignancies or lymphomas were recruited into this phase I, open-label, dose-escalation study of AZD8055 starting at 10 mg twice-daily oral dosing (BID). RESULTS: Forty-nine patients received AZD8055. Dose-limiting toxicities were reported at 40 mg (n=1), 90 mg (n=1) and 120 mg (n=3) BID; all were grade 3 rises in transaminases, reversible in all patients, apart from one who had liver metastases. The maximum tolerated dose was defined as 90 mg BID. The most frequent adverse events assessed to be related to AZD8055 were increased alanine aminotransferase (22%), increased aspartate aminotransferase (22%) and fatigue (16%). AZD8055 was rapidly absorbed (median t(max) ∼0.5 h) and exposure increased with increasing doses. Seven patients had stable disease for ≥ 4 months. Partial metabolic responses, assessed by fluorodeoxyglucose positron emission tomography, were observed at ≥ 40 mg BID (n=8 at day 35). CONCLUSION: The maximum tolerated dose for AZD8055 is 90 mg BID. Apart from elevated transaminases, which occurred at most dose levels, the drug had an acceptable toxicity profile; however, no RECIST responses were seen.

Recombinant human interleukin 4 has antiproliferative activity on human tumor cell lines derived from epithelial and nonepithelial histologies.

Interleukin 4, a T cell-derived 20-kDa glycoprotein, plays an important role in regulating the immune response of B cells, T cells, and macrophages against infections and malignant cells. For this reason recombinant human interleukin 4 (rhIL-4) has entered early clinical trials in cancer patients. In the present study we report that rhIL-4 has an antiproliferative effect on five of nine cell lines derived from human colon tumors, head and neck tumors, and glioblastomas as measured by a decrease of colony formation in human tumor cloning assays. All of the cell lines with in vitro responsiveness express at least 100 high-affinity receptors for human interleukin 4 per cell on their cell surface, whereas the nonresponsive tumor cell lines lack expression of high-affinity receptors for human interleukin 4 on their cell surface. In the next series of experiments we have xenotransplanted some of the responsive cell lines into athymic nude mice. Subsequently, the animals were treated s.c. twice daily with 0.5 mg/m2 rhIL-4 or control vehicle for at least 12 days. There was a clear growth inhibition of these xenotransplanted tumors in the mice treated with rhIL-4. Histology of the tumors in both groups revealed no marked infiltration with murine hematopoietic and lymphocytic cells as evaluated by staining with a rat anti-mouse CD45 antibody. We conclude that rhIL-4 has a direct therapeutic activity on the growth of some human epithelial and nonepithelial tumor cell lines which, along with its regulatory function on hematolymphopoietic cells, makes this cytokine an interesting candidate for experimental tumor therapy.

Inhibition of proliferation and clonal growth of human breast cancer cells by interleukin 13.

We tested the influence of recombinant human interleukin (rhIL)-l3 and rhIL-4 on clonal growth of human breast cancer cell lines. rhIL-13 and rhIL-4 inhibited clonal growth of three of nine lines to approximately 50% of controls (ED50, 0.5 ng/ml). rhIl-13 reduced [3H]thymidine incorporation in all three cell lines: two showing a minor (84% and 83% of controls) and one showing a major response (25% of control). Both cytokines markedly reduced serum-induced G(0/1) exit (approximately 25% versus 60%). 125I-labeled interleukin (IL) 13 binding assays revealed high-affinity binding sites for IL-13 on two of the three responding cell lines (KD approximately 60 pM). (Y124D)IL-4 effectively antagonized all effects of rhIl-13 and rhIL-4, arguing for shared receptor components between them. However, neither rhIl-4 nor (Y124D) IL-4 could displace 125I-labeled IL-13 from binding, although unlabeled rhIL-13 effectively did so. Using reverse transcription-PCR, we studied the expression of the common gamma chain (gammac) in responding cell lines, putatively being shared between IL-4 receptor and IL-13 receptor; none of the three cell lines express gammac. In conclusion, we demonstrate antiproliferative effects of IL-4 and IL-13 on carcinoma cells which express IL-13 binding sites without participation of gammac.

Nerve growth factor stimulates clonal growth of human lung cancer cell lines and a human glioblastoma cell line expressing high-affinity nerve growth factor binding sites involving tyrosine kinase signaling.

The growth of a panel of 22 different human tumor, leukemia, and lymphoma cell lines was examined in a human tumor cloning assay in agar or methylcellulose and a tritiated thymidine uptake assay. The cultures were performed in the absence or presence of increasing concentrations (0.5-500 ng/ml) of nerve growth factor (NGF). The growth of 17 of the 22 cell lines was not significantly and reproducibly affected by NGF. There was minor (1.2-fold) but reproducible stimulation of clonal growth in one glioblastoma cell line (86-HG-39) by NGF, but in this cell line NGF induced no growth modulation in a tritiated thymidine uptake assay. However, clonal growth of another glioblastoma cell line (87-HG-31) and all three lung cancer cell lines tested (HTB 119, HTB 120, CCL 185) could be stimulated up to 3-fold by NGF with a dose-response relationship for the growth factor. Growth stimulation by NGF could be completely reversed by neutralizing anti-NGF antibody and by the tyrosine kinase inhibitor genistein. Evaluation of secondary plating efficiency revealed the stimulation of colony formation as representing self-renewal and not terminal differentiation. Reverse transcriptase-PCR experiments in the five responding cell lines showed expression of both low-affinity NGF receptor (glycoprotein 75) and c-trk transcripts on the mRNA level. Of the five responding cell lines, only 86-HG-39, the cell line with the lowest responsiveness, revealed low-affinity NGF receptor on the protein level; the other four cell lines with high responsiveness, including the three lung cancer cell lines, expressed no low-affinity NGF receptor as shown by fluorescence-activated cell sorter analysis and immunoprecipitation using the ME 20.4 antibody. Immunoprecipitation using anti-trk antibodies was negative in all five responding cell lines. However, binding studies with iodinated NGF showed only low-affinity binding on the 86-HG-39 cell line and only high-affinity binding on the high-responder cell lines CCL 185 and 87-HG-31. In summary, our data suggest that NGF can be operative in stimulation of clonal growth of malignant tumor cells. High-affinity but not low-affinity binding sites mediate signal transduction for clonal growth and signaling involves tyrosine kinase activity.

Simultaneous immunomagnetic CD34+ cell selection and B-cell depletion in peripheral blood progenitor cell samples of patients suffering from B-cell non-Hodgkin's lymphoma.

The reduction of residual tumor cells is one of the main targets of leukapheresis product (LP) processing. Immunomagnetic enrichment/selection of CD34+ progenitor cells (Baxter Isolex 300i) can achieve a reduction of contaminating B-cells of approximately 2-3 logs in B-cell non-Hodgkin's lymphoma patients. Specific release of the enriched CD34+ cells (stem cell releasing agent PR34+; Baxter) and the use of antibody-coated immunobeads targeted against B-cell markers (CD10, CD19, CD20, CD22, CD23, and CD37) during this procedure allows the GMP-like simultaneous capture of residual B cells within a closed system. This combination of two purging techniques enhances the B-cell depletion capacity up to 4.5 logs. By performing 10 clinical-scale purging procedures, we could show that the simultaneous immunomagnetic purging method is easy to perform and highly efficient. We evaluated B-cell log depletion by flow cytometry for cases with marker-positive cells detectable before and after the purging procedure. The mean reduction of B-cells in these cases was 3.5 logs; the mean CD34+ cell yield and purity were 47 and 92%. Using three LPs, we tested the procedure on a modified Baxter Isolex 300i device with software adaptations for this procedure (software version 2.0) in direct comparison with CD34+ cell selection only, using the former version (version 1.12). The CD34+ cell yield was 49% (40-54%) for the CD34+ cell selection and 51% (19-72%) for simultaneous double selection. The mean purity was 96% for CD34+ cell selection and 98% for simultaneous double selection. B-cell depletion was 1.9 logs for CD34+ cell selection, and after simultaneous double selection, the B-cell content was decreased by 3.7 log steps (P = 0.0495). Clinical application of double-purged cells has not prolonged the hematopoietic recovery times after high-dose therapy as compared with nonpurged peripheral blood progenitor cell autotransplants. In conclusion, we could show that the simultaneous double selection protocol developed leads to a highly increased B-cell purging efficacy when compared with CD34+ cell selection without any negative effects regarding CD34+ cell yield and engraftment times after high-dose therapy.

Recombinant human stem cell factor does exert minor stimulation of growth in small cell lung cancer and melanoma cell lines.

We have previously reported on the stimulation of clonal growth of a glioblastoma cell line by rhSCF (Berdel et al., Cancer Res 1992, 52, 3498-3502). Within an extensive screening programme of haematopoietic growth factor activity on malignant cells, the effects of rhSCF were further tested on the growth of 29 different human cell lines derived from a wide range of solid tumours, among them six lung cancers and five melanomas. RhSCF (0, 1, 10, 100 ng/ml) was tested in a human tumour cloning assay (HTCA) which reliably detects growth modulation of tumour cells by cytokines. Additionally, a tritiated thymidine uptake test was used. Growth of 27 of the 29 cell lines tested was not affected by rhSCF. However, growth of the small cell lung cancer (SCLC) cell line HTB 120 was slightly stimulated (1.5 fold that of controls), and that of the melanoma cell line MeWo was stimulated up to 1.3-fold. This activity was eliminated dose-dependently by the tyrosine kinase inhibitor, genistein. We further analysed the cell lines for expression of the proto-oncogene C-KIT and its ligand SCF. All melanoma and lung cancer cell lines expressed SCF as assessed at the mRNA level. Northern blotting also revealed clear C-KIT mRNA expression in three melanoma (HAS, MeWo, SK-MEL-28), one NSCLC (HTB 53), and four SCLC cell lines (HTB 119, HTB 120, HTB 171, HTB 175). Furthermore, C-KIT protein expression was detected by flow cytometric analysis on the cell surface of MeWo, HTB 119 and HTB 120 cells. Our data indicate that SCF can be operative in growth modulation of non-haematopoietic malignant cells, especially SCLC and melanoma. However, our extensive screening of SCF/tumour cell interaction shows that this interaction is rare and makes potential hazards, such as tumour stimulation upon clinical use of rhSCF in conjunction with chemotherapy in cancer patients, unlikely for the majority of other tumour histologies.

Interleukin-1 receptor antagonist inhibits growth modulation of human tumor-cell lines by interleukin-1 in-vitro.

In this study we report that recombinant human (rh) interleukin-1alpha (IL-1alpha) has direct and dose-dependent growth-modulating effects on human tumor cell lines in vitro as measured by a human tumor cloning assay (HTCA). Colony formation of the melanoma cell line A 375 was inhibited by rhIL-1alpha, whereas colony formation of the glioblastoma cell line HTB 14 was enhanced by this cytokine. Both growth-modulating effects were dose-dependent, however with some saturation. Subsequently, we have tested the activity of recombinant human IL-1 receptor antagonist (rhIL-1ra) on the tumor growth modulation by rhIL-1alpha. Tumor cells were incubated with increasing concentrations of rhIL-1ra and then added to the cultures containing rHIL-1alpha. Concentrations of rhIL-1ra were chosen to achieve a range between 0.01 and 100 ng/ml which includes a 1000-fold molar excess over IL-1alpha. The receptor antagonist was able to block both the inhibition and the stimulation of clonal growth of the respective tumor cell line by rhIL-1alpha. Furthermore, there was a direct dose dependent relationship revealing higher IL-1 antagonism of rhIL-1ra at higher concentrations with maximum efficacy at 1000-molar excess concentrations over IL-1alpha. In addition, rhIL-1ra alone did not reveal major modulation of the growth of A 375, but significantly decreased colony formation of HTB 14. We conclude that rhIL-1ra can counteract modulation of clonal growth of human tumor cells by IL-1alpha in vitro. Since our report provides first evidence that the stimulation of clonal tumor cell growth by IL-1alpha can be blocked by rhIL-1ra, this member of the IL-1 cytokine network should be further studied as a possible candidate for experimental cancer treatment.

Fresh peripheral blood mononuclear cell preparations are a better starting material than bone marrow after cryopreservation for immunomagnetic harvesting of CD34(+) hematopoietic cells.

Immunomagnetic separation using anti-CD34 monoclonal antibodies and paramagnetic microspheres has been used to enrich hematopoietic stem cells from human bone marrow, whole cord blood, or mobilized peripheral blood mononuclear cell collections. This method has been reported to achieve high separation purity of CD34+ cells in small scale experiments with fresh material. The aim of the present study was to compare the efficacy of the CD34+ cell selection technique, when thawed bone marrow or fresh peripheral blood mononuclear cells were enriched. Starting with thawed bone marrow containing 2.9% CD34+ cells the final product purity was 67.7% with a 6% CD34+ cell yield (enrichment factor 25.7), and a 85-fold CFU-GM enrichment. Using fresh mobilized peripheral blood mononuclear cells the released cells contained 77.6% CD34+ cells with a 47% yield (enrichment 86.5-fold), and a 46-fold CFU-GM enrichment. These results indicate that CD34+ cells can be selected from cryopreserved bone marrow using immunomagnetic procedures. However, fresh leukapheresis products seem to be a much better material for a positive immunomagnetic stem cell selection technique in terms of purity, yield and enrichment.

Antiproliferative effect of human interleukin-4 in human cancer cell lines: studies on the mechanism.

Interleukin-4 (IL-4) plays an important role in activating the immune system against malignant cells. The human interleukin-4 receptor (hIL-4R) is not only expressed by hematopoietic cells but also on a large number of tissue specimens which include colon, breast and lung carcinomas. In this study we report that rhIL-4 has an antiproliferative effect on 2 out of 3 non-small cell lung carcinoma (NSCLC) cell lines in vitro as measured by human tumor cloning assays (HTCA). In comparison, rhIL-4 had no effect on the growth of small cell lung carcinoma cell lines (SCLC) in vitro. The response towards the cytokine is correlated with expression of at least 1500 high affinity receptors/cell for hIL-4 on the responsive cell lines. Xenotransplanting the human lung tumor cell lines into nude mice followed by 12 days of systemic treatment of the mice with rhIL-4 revealed a significant growth retardation of the IL-4R positive NSCLC cell lines when compared with the controls, whereas the growth of the IL-4R negative SCLC cell lines was unaffected also in vivo. Studies of possible mechanisms involved in the antiproliferative effect of rhIL-4 showed that rhIL-4 does not induce apoptosis or modulation of the transcription factor c myc in the responsive NSCLC cell lines. Additionally, the expression of the epidermal growth factor receptor (EGFR), which is discussed as mediating autocrine/paracrine growth stimulation of NSCLC, is unaffected by rhIL-4. However, we have observed that rhIL-4 inhibited G1-S-phase cell cycle progression. We conclude that rhIL-4 has an antiproliferative effect on the growth of some NSCLC in vitro and in vivo. The mechanisms involved remain to be further elucidated.

Early tandem high-dose ifosfamide, carboplatin, etoposide therapy with stem cell rescue for small-cell lung cancer: brief report on the results of a phase-I/II trial.

OBJECTIVE: High-dose therapy (HDT) for small-cell lung cancer is experimental. Late intensification HDT for chemosensitive disease can increase the number and quality of remissions and prolong relapse-free survival, but has not yet shown impact on overall survival. This is possibly due to resistant residual disease. To overcome the development of resistance, we have tested early intensification tandem HDT. METHODS: We performed a phase-I/II trial using 1 conventional cycle of ifosfamide, carboplatin, etoposide (ICE) plus granulocyte colony-stimulating factor for stem cell recruitment followed by 2 cycles of high-dose ICE with rescue by CD34+ cell-enriched peripheral blood mononuclear cells. Dose escalation was performed for the 2 high-dose ICE cycles. Radiotherapy for limited disease was according to standard protocols. RESULTS: 17 patients were entered: 2 female patients; 15 male patients; median age 53 (range 36-65) years; 2 patients with limited disease, and 15 patients with extensive disease. We treated 4 patients at dose level 1, 11 patients at level 2, and 2 patients at level 3. The maximum tolerable dose was at level 2 with neuropathy being dose-limiting. Overall, toxicity was < or = grade 2 for all patients up to dose level 2 with hematotoxicity being grade 4 for all patients. There were 15 partial remissions (88%), 1 no change (6%), and 1 progressive disease (6%). Median time to progression was 7.9 months. Overall survival was 12.9 months (median). CONCLUSIONS: Early intensification with this protocol is feasible. Although a comparatively good response rate and median time to progression have been observed in this group dominated by patients with extensive disease, overall survival is short and no substantial long-term survival was found.

Autocrine interleukin-1 receptor antagonist can support malignant growth of glioblastoma by blocking growth-inhibiting autocrine loop of interleukin-1.

In situ hybridization (ISH) of human glioblastoma tissue sections revealed expression of interleukin-1 (IL-1)alpha and/or beta and IL-1 receptor types I and II (IL-1R I and II) in the majority of cases evaluable. To understand the function of IL-1-family members in human glioblastomas, we have studied 6 glioblastoma cell lines. RT-PCR, ISH, ELISA and 125I-IL-1-binding assays revealed expression of IL-1 and high-affinity receptors for human (h)IL-1 in all but 1 cell line. Using a colony growth assay in semi-solid media for testing serial plating efficacy (PE, number of colonies per number of cells seeded in %), only the IL-1R-negative cell line was not influenced by recombinant human (rh)IL-1alpha or -beta, whereas IL-1 down-regulated the self-renewal of clonogenic cells of the other glioblastomas. Tritiated thymidine uptake was down-regulated by rhIL-1 in all cell lines studied. Cell viability remained unchanged by rhIL-1. Wherever growth modulation by rhIL-1 was detected, it could be reversed by either soluble IL-1R I or II or by rhIL-1 receptor antagonist (ra). IL-1ra not only was able to reverse rhIL-1-induced growth modulation but alone could modulate glioblastoma growth in comparison with control in cell lines producing IL-1. Our results show the presence of public autocrine loops for IL-1 leading to growth inhibition in some glioblastomas. To understand these loops, we have studied expression and function of IL-1ra in glioblastomas. ISH of human glioblastoma tissue sections revealed expression of hIL-1ra in all 8 cases evaluable. In 4 of 6 cell lines, IL-1ra was found in the supernatant under constitutive conditions, the IL-1R-negative line being among the 2 non-producers. The other non-producing cell line, HTB 17, showed expression of hIL-1R II. Most interestingly, a neutralizing antibody against IL-1ra down-regulated growth of IL-1- and IL-1ra-producing glioblastoma cells to approx. 30% of the controls. Thus, public autocrine loops for IL-1 in human glioblastomas exist and result in growth inhibition. An autocrine production of IL-1-antagonizing molecules such as IL-1ra by these tumors can counteract this IL-1 function and represent a basic escape mechanism supporting malignant growth in some glioblastomas.

Lymphotoxin, tumour necrosis factor and interleukin-6 gene transcripts are present in Hodgkin and Reed-Sternberg cells of most Hodgkin's disease cases.

Tissue specimens from 26 cases of Hodgkin's disease (HD) and six HD-derived cell lines were analysed for tumour necrosis factor (TNF), lymphotoxin (LT), and interleukin (IL)-6 RNA transcripts by in situ hybridization, in some cases subsequent to immunohistology for CD30 antigen. LT and TNF transcripts were found in tumour cells of all cases; IL-6 gene transcripts were detectable in 19/23 cases. Presence of RNA specific for these cytokines was not correlated with any of the following parameters: sex, symptoms and histotype, as well as immunophenotype and association of the tumour cells with Epstein-Barr virus. Rather, the presence of LT, TNF and IL-6 transcripts appeared to characterize Hodgkin and Reed-Sternberg cells in general, supporting concepts which suggest that HD represents a malignancy of cytokine secreting activated cells, and that many of the features distinguishing HD from other malignant lymphomas may ultimately be due to expression of cytokines. LT and TNF RNA transcripts were also found in five HD-derived cell lines, whereas supernatants of these cell lines contained high levels of LT, but low or undetectable levels of TNF activity. This suggests that, although not detectable at the level of RNA transcripts, differences between HD cases may exist on the level of cytokine gene transcript processing, translation and polypeptide secretion.

Immunomagnetic selection of CD34+ cells from fresh peripheral blood mononuclear cell preparations using two different separation techniques.

Immunomagnetic separation using anti-CD34 monoclonal antibodies and paramagnetic microspheres has been used to enrich hematopoietic stem cells from human bone marrow, whole cord blood, or mobilized peripheral blood mononuclear cell collections. The aim of the present study was to compare the efficacy of two different CD34+ cell selection techniques in enriching CD34+ cells from mobilized fresh peripheral blood mononuclear cells. Using the magnetic cell sorter (MACS), the final product purity was 74.1% CD34+ cells (starting population 2.3% +/- 3.3%) with a 60.3% CD34+ cell yield. Using Dynabeads and subsequent chymopapain incubation for releasing the target cells from the beads (Isolex system), the released cells contained 83.3% CD34+ cells (starting population 1.2% +/- 0.7%) with a 43.4% yield. These results indicate that CD34+ cells can be isolated with high purity from fresh leukapheresis products using both immunomagnetic techniques.

Constitutive activation of FLT3 in acute myeloid leukaemia and its consequences for growth of 32D cells.

The receptor tyrosine kinase Flt3 is expressed on leukaemic blasts of most cases with acute myeloid leukaemia (AML). In order to evaluate the presence and significance of constitutive activation of Flt3 for leukaemogenesis, we (1) analysed the expression and activation status of the receptor in AML blasts; and (2) evaluated the functional consequences of constitutively active Flt3 in a myeloid progenitor cell line. Immunoprecipitation studies revealed Flt3 expression in a high proportion of AML cases (27/32) with ligand-dependent Flt3 autophosphorylation in 18, constitutive autophosphorylation in three and no autophosphorylation in six cases. Only one out of three samples with constitutively active Flt3 but 3/18 samples with ligand-dependent autophosphorylated Flt3 contained the recently described internal tandem repeat (ITR) mutations. To test the significance of Flt3 activation in myeloid cell function, we also characterized the biochemical and biological effects of the activating mutation D838V of Flt3 (FLt3D838V) on the factor-dependent myeloid progenitor cell line 32Dcl3: cells transfected with wild-type Flt3 (32D/Flt3) grew FLt3 ligand (FL) dependent, and the receptor was ligand dependently autophosphorylated. In contrast, the receptor was constitutively autophosphorylated in 32D/Flt3D838V cells, which grew independently of FL. We conclude that, in some AML samples, Flt3 is constitutively activated and that this does not correlate with ITR mutations in the juxtamembrane domain. Furthermore, constitutively active Flt3 confers factor independence to the myeloid progenitor cell line 32D. It remains to be determined whether activation of Flt3 is leukaemogenic in vivo and whether strategies aimed at inhibition of Flt3 activation could inhibit leukaemogenesis.