Granulocyte colony-stimulating factor given in addition to interferon-α to mobilize peripheral blood stem cells for autologous transplantation in chronic myeloid leukaemia

In order to potentially mobilize and harvest the Ph⁻ cells observed in most patients with chronic myeloid leukaemia (CML) during interferon-α (IF-α) therapy, G-CSF (filgrastim), 5 μg/kg/d, was administered subcutaneously together with IF-α to 30 CML patients in haematological remission but with various degrees of cytogenetic remission, after IF-α therapy. Peripheral blood stem cells (PBSC ) were harvested using standard aphereses from day 5 of G-CSF. Patients underwent one to four (median three) aphereses. Median total yields/kg were 7.6 (range 3.8–25) × 10⁸ MNC, 3.4 (0–140) × 10⁶ CD34⁺ cells, and 17 (1.1–107) × 10⁴ CFU-GM. No patient had a significant increase in the percentage of Ph⁺ cells in the bone marrow under G-CSF therapy. The percentage of Ph⁺ cells in apheresis products tended to decrease between the first and the last apheresis ( P  = 0.05). 14 patients who were not responsive to IF-α were transplanted after conditioning with busulphan 16 mg/kg and melphalan 140 mg/m². Median time to neutrophils > 0.5 × 10⁹/l was 20 d (16–114 d) and to platelets > 50 × 10⁹/l 18 d (12–149 d). Nine patients had a major cytogenetic response post graft, which correlated with the amount of Ph⁺ cells reinfused with the graft ( P  = 0.02). We conclude that this procedure is feasible, allowing the harvest of enough PBSC, some of them Ph⁻ in patients who responded to IF-α, to allow autologous transplantation.     

Autologous transplantation in chronic myeloid leukaemia using peripheral blood stem cells

Forty-three patients with chronic myeloid leukaemia in first chronic phase were recruited to study intensive chemotherapy (idarubicin plus cytarabine; IdAC) followed by collection of peripheral blood stem cells (PBSC) in the recovery phase. PBSC autografting was performed on 32 patients. One patient died during mobilization and three died following autograft. All procedural deaths occurred in patients who received IdAc more than a year from diagnosis. Nine further patients died, eight following progression of CML. 72% of transplanted patients showed a major cytogenetic response but most cases have returned to Philadelphia-positive haemopoiesis. 62% of autografted patients remain alive (median survival from diagnosis 52 months). Four of the 11 patients who did not receive a transplant remain in chronic phase.     

Effect of interferon-α on immunoglobulin production by peripheral blood mononuclear cells in multiple myeloma

Abstract: To test a hypothesis that interferon-α (IFN) treatment might restore normal immunoglobulin (Ig) production in multiple myeloma (MM), the effect of IFN on Ig isotype (IgG and IgA) production by peripheral blood (PB) and bone marrow (BM) mononuclear cells (MNCs) in MM patients was analyzed by ELISA. IFN at a concentration of 1000 U/ml was found to enhance IgA production by PB MNCs in IgA-MM and had a trend to stimulate IgG production in IgG-MM. The effect of IFN on nonparaprotein Ig isotype production was more variable, with mostly neutral or inhibitory effects being seen in both the MM subtypes. In contrast to the influences observed in MM patients, IFN at the same concentration inhibited both IgG and IgA production by PB MNCs in healthy controls. In studying BM cells, IFN was found to reduce IgA production in IgA-MM, but had a neutral effect on IgG production in IgG-MM. In the controls, the production of both the IgG and the IgA isotypes by BM MNCs was decreased by IFN. On the basis of these results it seems that the disease itself somehow affects the Ig-producing cells in MM, when measured as different responses of the cells to exogenous IFN in vitro. The results do not support the hypothesis that IFN treatment could restore normal Ig production in MM patients.     

Cutaneous sarcoidosis in a patient with philadelphia-positive chronic myelogenous leukemia treated with interferon-α

A patient with Philadelphia-positive (Ph+) chronic myelogenous leukemia (CML) was diagnosed with cutaneous sarcoidosis after treatment with interferon-alpha (IFN-α). Following IFN-α dose reduction, the skin lesions disappeared. Few cases of sarcoidosis associated with IFN treatment have been reported, and only in one patient with pre-existing CML. Our patient was unique in that (1) the sarcoidosis was induced by the IFN-α treatment alone, (2) it developed de novo, and (3) it was confined to the skin. Am. J. Hematol. 58:80–81, 1998. © 1998 Wiley-Liss, Inc.     

Changes in adhesion molecule expression and function in B-cell chronic lymphocytic leukaemia after in vitro interferon-α stimulation

Abstract: Peripheral blood mononuclear cells (PBMCs) from 10 B-CLL patients were investigated after 24 hours of in vitro interferon-α (IFN-α) stimulation. The constitutional expression of the L-selectins (LECAM-1), LFA-1/CD11a, VLA α-4/CDw49d and ICAM-1/CD54 adhesion molecules was detected, and changes in their density after IFN-α stimulation were compared to results obtained by the high endothelial venule (HEV)-binding assay and a carbohydrate (phosphonomannan core polysaccharide: PPME and fucoidin) immobilization test. The LECAM-1 and ICAM-1 molecules were expressed on the great majority of CLL cells, while the LFA-1 and VLA-4 α-chains were expressed by only a small number of cells. Statistically significant changes (p< 0.001) were observed in LECAM-1 antigen density (changes in mean cell fluorescence), as well as in functional tests (HEV-, PPME- and fucoidin-binding; p<0.01) after in vitro IFN-α stimulation. Based on a prior study (Jewell et al., Leukemia 1992: 6: 400–404) and on the present findings, not only an increased expression but also an enhanced function of the L-selectins seem to be well substantiated after IFN-α stimulation, which may explain the therapeutic effect of IFN-α in reducing the accumulation of leukaemic B cells in the blood. The remarkably high expression of ICAM-1 in this series necessitates further studies to clarify the exact expression rate and role of this molecule.     

b3a2 BCR-ABL fusion peptides as targets for cytotoxic T cells in chronic myeloid leukaemia

Peptide sequences spanning the BCR-ABL protein junction potentially constitute novel leukaemia-specific antigens. 9-mer b3a2 fusion peptides have been reported to bind with high affinity to HLA-A3, -A11 and -B8. We have studied the effect of b3a2 BCR-ABL junctional peptides on the cytotoxic T-cell (CTL) response against normal and chronic myeloid leukaemia (CML) cells. Antigen-presenting cells (APCs) were prepared from HLA-A3- or -B8-positive peripheral blood mononuclear cells (PBMCs) by incubation with phytohaemagglutinin (PHA) and interleukin (IL)-2 for 7 d. These APCs were pulsed with the respective b3a2 junctional peptide in the presence of beta2-microglobulin and were then used to challenge autologous PBMCs at 7-d intervals in the presence of IL-2, IL-6, IL-7 and IL-12. On subsequent exposure to target cells (either further pulsed normal APCs or unpulsed CML cells), specific HLA-restricted CTL responses were observed against all HLA-A3/-B8 matched normal target cells tested, but not to targets that were HLA mismatched. Cytotoxicity was also induced against HLA-A3/-B8 unpulsed CML cells, but not against unmatched CML cells. These data indicate (i) that endogenous BCR-ABL junctional peptides may be presented by CML cells and (ii) that exogenous peptides are potential stimulators of autologous antileukaemic CTLs.     

A double-blind controlled trial of recombinant interferon-α2b in haemodialysis patients with chronic hepatitis C virus infection and abnormal aminotransferase levels

SUMMARY. The efficacy and safety of recombinant interferon-α2b (rIFN-α2b) was evaluated in a double-blind controlled trial comprising 23 haemodialysis patients with antibodies to hepatitis C virus (anti-HCV), detectable serum HCV RNA by polymerase chain reaction and chronic alanine aminotransferase elevation. The patients were randomly assigned to receive rIFN-α2b at a dose of 1.5 MU (increasing to 3 MU if no response was observed) (Group I: n = 14) or identical placebo (Group II: n = 9), for 6 months. A biochemical response (normal alanine aminotransferase) was observed in 10 patients (71.4%) from Group I and in one patient (11.1%) from Group II (P < 0.01) at the end of therapy, and in four patients from Group I (28.6%) and in none from Group II (NS) 12 months after therapy. Virological response (HCV RNA negative) was observed in four patients (28.6%) from Group I and in none from Group II (NS) at the end of therapy, and in two patients (14.2%) from Group I and in none from Group LT (NS) 12 months after therapy. Interferon doses were 1.5 MU in 12 patients and 3 MU in two patients. Therapy interruption owing to severe side-effects was necessary in three patients (21.4%) from Group I and in two patients (22.2%) from Group II. Although long-term statistical differences were not observed, these results suggest that rIFN-α2b at a low dose is a reasonable and well tolerated therapeutic approach for haemodialysis patients with chronic hepatitis C.     

Treatment of chronic myeloid leukaemia in first chronic phase with idarubicin and cytarabine: mobilization of Philadelphia-negative peripheral blood stem cells

Peripheral blood stem cell (PBSC) mobilization using idarubicin and cytarabine was investigated in 40 patients with chronic myeloid leukaemia in first chronic phase (CML CP1). Disease contamination was evaluated in harvests from 41/44 (93%) mobilization episodes. Using cytogenetics, 22/37 (59%) showed a complete or major response; Southern blot analysis demonstrated a complete or major response in 9/17 (53%). No harvests were RT-PCR negative. In the 41 evaluable episodes, more complete or major responses were seen when PBSC mobilization occurred within 24 months [17/23 (74%) versus 6/18 (33%); P  =0.02] and within 12 months of diagnosis [10/11 (91%) versus 13/30 (43%); P  = 0.018]. 20 patients underwent PBSC transplantation and 18/20 successfully engrafted. Post-transplant cytogenetic analysis was available on 15 cases, of whom five achieved a major cytogenetic response at 1–3 months, with five partial cytogenetic remissions. Two of 40 patients died during mobilization therapy (5%) and three of 20 after the transplant (15%). Overall mortality was high at five of 40 patients, and the procedural mortality was 20%. This study demonstrates that Ph-negative PBSCs can be mobilized in a significant proportion of patients with CML CP1, with the best results observed within a year of diagnosis. These cells can subsequently be used for autologous transplantation, however, the impact on long-term survival requires longer follow-up, and potential benefits may be compromised by the high mortality.     

Neutralizing anti-interferon-α antibodies and response to treatment in patients with Ph+ chronic myeloid leukaemia sequentially treated with recombinant (α2a) and lymphoblastoid interferon-α

Neutralizing anti-IFNα antibodies (nIFNα Abs) occur in a significant proportion of patients with hairy cell leukaemia, hepatitis or solid tumours treated with recombinant IFNα (IFNα2a or IFNα2b), but information on their incidence in chronic myeloid leukaemia (CML) is scanty and their clinical relevance is not yet completely defined. By using an IFNα antiviral neutralization bioassay, the frequency of nIFNα2a Abs was evaluated in 67 Ph⁺ CML patients during IFNα2a therapy at doses ranging from 6 to 9 MU/d. 15 patients (22%) developed nIFNα2a Abs (titre ranging from 1:40 to 1:20480) and 11/15 (73%) were haematologically and/or karyotypically unresponsive to treatment. 52 patients did not develop antibodies and 11 of them (21%) were unresponsive. The negative relationship between the positivity for nIFNα2a Abs and the response to treatment was highly significant (P = 0.0001). In nine nIFNα2a Abs positive patients, treatment was changed from recombinant IFNα2a to lymphoblastoid IFNα (IFNα-ly), at the same dose and schedule. After 9 months of IFNα-ly treatment a haematological response was achieved in 4/7 cases who were non-responsive to prior IFNα2a therapy and was maintained in the other two patients previously responsive to IFNα2a. However, no karyotypic response was observed. This data shows that a significant proportion of Ph⁺ CML patients receiving treatment with IFNα2a can develop neutralizing antibodies and that these antibodies are associated with a loss of IFNα2a efficacy. Changing the patients to treatment with lymphoblastoid IFNα may restore haematological response but it is not likely to induce a karyotypic response.     

Hyperglycaemia due to insulin resistance caused by interferon-γ

Marked hyperglycaemia (30.9 mmol l⁻¹) during interferon-γ (IFN-γ) therapy for asymptomatic recurrent renal cancer as multiple lung metastases in a 52-year-old man is described. Although the involvement of IFN-γ has been reported in the development of autoimmune diabetes, in this case, antibodies against pancreatic β-cells including anti-islet cell antibody (ICA) and anti-glutamic acid decarboxylase (GAD) antibody were negative. Moreover, serum level of immunoreactive insulin (IRI) (11 μU ml⁻¹ at fasting) and urinary excretion of C-peptide (108 μ g day⁻¹, reference range: 20–130) suggested insulin resistance, supported by results of insulin tolerance tests. With insulin therapy and cessation of IFN-γ, fasting blood glucose concentration returned to 6.2 mmol l⁻¹, and insulin therapy was discontinued. The injection of IFN-γ may cause hyperglycaemia because of insulin resistance, rather than β-cell injury. © 1998 John Wiley & Sons, Ltd.     

Blood outgrowth endothelial cells from chronic myeloid leukaemia patients are BCR/ABL1 negative

The existence of adult haemangioblasts with dual haematopoietic and endothelial developmental potential was confirmed after detection of Ph⁺ vascular endothelial cells in chronic myeloid leukaemia (CML) patients. Blood outgrowth endothelial cells (OECs) from CML patients were found not to harbour the Philadelphia translocation and were thus not clonally related to BRC/ABL1 ⁺ hematopoietic progenitors, but comprised a distinct subfraction of endothelial cells. Remarkably, the frequency of CML-derived OECs was 9-fold higher as compared to healthy donors ( n  = 19 and n  = 300, respectively; P  <   0·0001) and these cells showed increased proliferative potential, possibly reflecting the mobilisation of OEC progenitors by pro-angiogenic cytokines.     

Factors influencing long‐term efficacy and tolerability of bosutinib in chronic phase chronic myeloid leukaemia resistant or intolerant to imatinib

The dual SRC/ABL1 tyrosine kinase inhibitor bosutinib is indicated for adults with Ph+ chronic myeloid leukaemia (CML) resistant/intolerant to prior therapy. This analysis of an ongoing phase 1/2 study (NCT00261846) assessed effects of baseline patient characteristics on long‐term efficacy and safety of bosutinib 500 mg/day in adults with imatinib (IM)‐resistant (IM‐R; n = 196)/IM‐intolerant (IM‐I; n = 90) chronic phase (CP) CML. Median treatment duration was 24·8 months (median follow‐up, 43·6 months). Cumulative major cytogenetic response (MCyR) rate [95% confidence interval (CI)], was 59% (53–65%); Kaplan‐Meier (KM) probability of maintaining MCyR at 4 years was 75% (66–81%). Cumulative incidence of on‐treatment progression/death at 4 years was 19% (95% CI, 15–24%); KM 2‐year overall survival was 91% (87–94%). Significant baseline predictors of both MCyR and complete cytogenetic response (newly attained/maintained from baseline) at 3 and 6 months included prior IM cytogenetic response, baseline MCyR, prior interferon therapy and <6 months duration from diagnosis to IM treatment initiation and no interferon treatment before IM. The most common adverse event (AE) was diarrhoea (86%). Baseline bosutinib‐sensitive BCR‐ABL1 mutation was the only significant predictor of grade 3/4 diarrhoea; no significant predictors were identified for liver‐related AEs. Bosutinib demonstrates durable efficacy and manageable toxicity in IM‐R/IM‐I CP‐CML patients.     

Phosphatidylinositol-3 kinase inhibitors reproduce the selective antiproliferative effects of imatinib on chronic myeloid leukaemia progenitor cells

We investigated the role of the phosphatidylinositol-3 kinase (PI-3K) pathway in regulating the proliferation of primary chronic myeloid leukaemia (CML) progenitor cells by using imatinib to inhibit the activity of p210(Bcr-Abl). The effect of imatinib on the expression of PI-3K pathway proteins was investigated by kinase assays and Western blotting; PI-3K was inhibited by wortmannin or LY294002, Jak2 by AG490 and farnesylation by FTI II; progenitor cell proliferation (self-renewal) was measured by growing myeloid colonies in vitro, then replating them to observe secondary colony formation. Suppression of p210(Bcr-Abl) with imatinib indirectly suppressed the activity of PI-3K and its downstream targets (Erk, Akt and p70S6 kinase), thereby implicating the PI-3K pathway in p210(Bcr-Abl)-mediated signalling in primary CML progenitor cells. The PI-3K inhibitors, wortmannin and LY294002 reproduced the differential effects of imatinib on normal and CML progenitor cell proliferation in vitro by increasing normal cell (P = 0.001) and reducing CML cell proliferation (P = 0.0003). This differential effect was attributable to dysregulated signalling by granulocyte colony-stimulating factor in CML. The responses of individual patient's cells to wortmannin correlated with their responses to imatinib (P = 0.004) but not their responses to AG490 (Jak2 kinase inhibitor) or FTI II (farnesyltransferase inhibitor). Individual responses to wortmannin also correlated with responses to interferon alpha (IFNalpha) (P = 0.016). Imatinib-resistant K562 cells were sensitive to LY294002. Inhibition of the PI-3K pathway may be common to imatinib and IFNalpha and reflect dysregulated cytokine signalling. As imatinib-resistant cells remained sensitive to wortmannin and LY294002, targeting the PI-3K pathway may provide an alternative therapy for imatinib-resistant patients.     

Molecular remission of chronic myeloid leukaemia following a non-myeloablative allogeneic peripheral blood stem cell transplant: in vivo and in vitro evidence for a graft-versus-leukaemia effect

Two patients with chronic myeloid leukaemia (CML) received a non-myeloablative preparative regimen of cyclophosphamide and fludarabine, followed by an unmanipulated, G-CSF-mobilized, peripheral blood stem cell transplant from an HLA-identical sibling. Chimaerism, evaluated in myeloid and T-lymphoid lineages by PCR of minisatellite variable regions, showed day 14 post-transplant haemopoietic recovery to be 90% autologous in both patients. On day 30 the bone marrow showed only 1/20 and 2/18 donor metaphases. By day 100 post transplant both had 100% donor myeloid and lymphoid lineages as assessed by karyotype and minisatellite chimaerism analysis. They subsequently became RT-PCR negative for BCR-ABL. Both survive 7 and 14 months post transplant in molecular remission of CML. In one, donor T cells, stimulated with pre-transplant CML cells, induced 30-50% inhibition of pre-transplant leukaemic CFU-GM, but did not inhibit CFU-GM in the day 60 marrow (46% Ph-negative recipient, 54% donor). These results show that a non-myeloablative allotransplant can induce molecular remissions of CML through a graft-versus-leukaemia effect.