ALBERT

Description: Point mutations within the BCR-ABL kinase domain represent the most common mechanism of resistance to imatinib in patients with CML. Preclinical studies have shown that dasatinib (BMS-354825) is effective at inhibiting the kinase activity of imatinib-resistant BCR-ABL mutants with the notable exception of the T315I mutation, which remains highly resistant to imatinib, dasatinib, and AMN107 (Gorre et al, Science 2001; Shah et al, Science 2004; Weisberg et al, Cancer Cell, 2005). Clinical data from Phase I and II studies of dasatinib in CML confirms the in vitro findings. Each of three imatinib-resistant patients bearing the T315I mutation (CP=1; AP=2) did not achieve objective hematologic or cytogenetic responses during treatment with dasatanib on a Phase I study. Additionally, each of two phase II patients with the T315I mutation (CP=1; LBC=1) treated at UCLA showed no evidence of objective response. We have also detected the T315I mutation in each of two cases of acquired resistance in a phase II (LBC =2) study, and in seven of nine patients with acquired resistance to dasatinib in phase I and II studies (CP=1; MBC=3; LBC=2; Ph+ ALL=1). Notably, we detected a novel BCR-ABL mutation, T315A, in one of the two patients who relapsed without a detectable T315I mutation. The patient is a 53 year-old female whose chronic phase CML had progressed to myeloid blast phase while being treated with imatinib. The imatinib-resistant mutation M244V was identified prior to dasatinib treatment. The patient achieved a major hematologic response (

Description: Imatinib mesylate (IM) has dramatically changed the treatment approach for patients with chronic myeloid leukemia (CML). However, ~20% of chronic phase (CP) patients are initially resistant to IM, and among patients who achieve a complete cytogenetic response (CCyR), a small minority relapse back into CP or progress to advanced disease. Abl tyrosine kinase domain mutations are the major cause of secondary resistance. Oligonucleotide microarray analysis was used to study gene expression patterns associated with primary IM resistance and relapse after initial successful response to IM. Samples included total RNA from diagnostic samples of 25 CML CP patients within 6 months of diagnosis and at the time of failure (7 patients); total RNA from 18 patients who relapsed after initial CCyR with documented Abl point mutations (12 CP and 6 BC); and 10 myeloid progenitor samples (sorted by CD34 and CD38 status) from an IM naïve and IM resistant (R) patient, both in blast crisis. Results: For primary IM resistance, analysis of paired samples before and after treatment revealed that primary failure was associated with the differential expression of genes associated with RNA post-transcriptional modification, protein synthesis, cellular growth and proliferation, and cell death. Two genes with the highest differential expression included the apotosis resistance genes API5 and TRAF5. TRAF5 was also increased in patients who relapsed after initial CCyR, while API5 showed significantly increased expression in sorted CD34+ cells from an IMR patient. In secondary resistance patients, a set of drug transporters including ABCA2, ABCA3, MDR1, and ABCC3 had increased expression and hOCT1 decreased expression relative to 42 IM naïve CP patients. In vitro experiments compared K562 resistant (R) and sensitive (S) cell lines over time exposed to IM. The K562 IMR cell line showed a 1.5 log higher expression of ABCG2 and a 1 log higher expression of TRAF5 compared to the K562 IMS cells. However, sequential clinical samples of 16 IM non-responders vs. 14 CCyR patients showed no change in ABCG2 expression, possibly because ABCG2 is expressed only in early progenitor cells, not differentiated cells. The importance of using CD34+ cells was demonstrated in 7 array studies comparing gene expression in CD34+ selected cells from an IMR patient compared to an IM naïve patient. IM resistance was associated with increased expression of genes associated with cell cycle, DNA recombination and repair, and proliferation. Genes associated with apoptosis resistance included increased expression of API5, survivin, and decreased expression of BAK1. Protein serine/threonine and tyrosine kinases associated with cell proliferation/survival and tumor progression with increased expression in the IMR patient included: AURKB, AKT3, BUB1B, CDC2, CHEK1, MAPK9, STK6, TTK, and WEE1. Conclusion: Gene expression studies suggest that primary resistance to IM therapy is associated with resistance to apoptosis; relapse on IM is associated with activation of drug transporter genes and genes associated with disease progression; in studying disease response and resistance, primitive hematopoetic cells are critical for analysis.

Description: The gastrointestinal tract is the commonest extra-nodal site of involvement in B-cell lymphomas and poses particular diagnostic and management problems, including therapy induced bowel perforation and bleeding. Rituximab therapy has demonstrated a significant increase in the complete response rate and prolongs disease free (DFS) and overall survival (OS) in diffuse large B-cell lymphoma. The extra efficacy of rituximab based therapies might increase the immediate complications peculiar to lymphomas of the gastrointestinal tract, particularly perforation. This has not been studied previously. We have reviewed treatment related complications and outcome in 40 cases of primary gastric and intestinal B-cell lymphoma (36 Diffuse Large B-Cell lymphoma, 3 Burkitt Lymphoma, 1 post transplant lymphoproliferative disorder) of the gastrointestinal tract (esophagus 1 case, stomach 21, small bowel 14, ileocaecal 3, large bowel 7;some patients had more than one site involved) over a 25 year period. Rituximab therapy was added to conventional therapy for patients treated in the last 5 years. There were 10 patients (median age 60; range 35–79; M: F=1.5:1) treated with regimens that included rituximab and 30 patients (median age 56; range 12–91; M: F=1.5:1) treated without rituximab. This included chemotherapy (12 patients), surgery alone (2 patients), both surgery and chemotherapy (14 patients) and radiotherapy (1 patient). One patient was not fit for any type of therapy. Complications occurred in 7 patients not treated with rituximab and in 1 patient who received this treatment. Complications included perforation (3 patients treated without rituximab; 1 patient in the rituximab group), bleeding (3 patients treated without rituximab; no episodes of bleeding in the rituximab group) and intestinal obstruction (3 patients in the group not treated with rituximab; no episodes of obstruction in the rituximab treated group). The difference in the complication rate between the two groups was not significant. Five patients in the group that did not receive rituximab died; two of neutropenic sepsis, two of gastric perforation and the other of operative complications. There were no treatment related deaths in the rituximab group. There was one patient in the rituximab treated group and three patients in the group that did not receive rituximab who had refractory disease. The median DFS was 21 and 14 months in patients treated with and without rituximab, respectively (p=0.6). The mean OS was 35 months in the rituximab treated group and 45 months in the group that did not receive rituximab (p = 0.9). There is presently no evidence that rituximab should be withheld early in the treatment of primary gastro-intestinal B-cell lymphomas because of concern about increasing the complication rate.

Description: The human α globin cluster includes an embryonic gene ζ and 2 fetal/adult genes (α2 and α1) arranged along the chromosome in the order in which they are expressed in development (5′-ζ-pseudoζ- αD- α2-α1-?-3′). Fully activated expression of these genes in erythroid cells depends on upstream regulatory elements of which HS-40, located 40kb upstream of the cluster, appears to exert the greatest effect. We have recently shown that during terminal differentiation, key transcription factors (GATA-2, GATA-1, NF-E2, SCL complex) sequentially bind the α promoters and their regulatory elements and a domain of histone acetylation develops which eventually encompasses the entire α globin cluster including the upstream regulatory sequences. α-thalassemia most frequently results from deletions or point mutations affecting the structural α globin genes, but may also result from rare sporadic deletions which remove the upstream regulatory sequences. In a single family α globin expression was silenced by a mutation which drives an anti-sense RNA through the α gene. Alpha thalassemia may also result from inherited and acquired mutations in a trans-acting factor called ATRX. Over the past few years we have continued to screen for new mechanisms which lead to α thalassemia and thereby elucidate new principles underlying the regulation of gene expression in hemopoiesis. Here we describe a new mechanism of α thalassemia occurring in Pacific Islanders in whom we could detect no mutations or rearrangements in the α globin gene locus. Despite this, extensive genetic analysis showed unequivocally that the causative mutation is linked to the terminal 169kb of chromosome 16 (Viprakasit et al accompanying abstract). Analysis of globin synthesis, steady state RNA levels and detection of RNA in situ demonstrated that the mutation downregulates α globin transcription. To identify the mutation, we constructed a new BAC library from an affected homozygote, isolated and re-sequenced the candidate region and focussed further analysis on 8 SNPS within the α globin cluster, one of which creates a new GATA-1 binding site (GACA〉GATA). Using primary erythroblasts from normal individuals and patients with this form of thalassemia, together with interspecific hybrids containing either the normal or abnormal copy of chromosome 16, we have shown that this SNP creates a new binding site in vivo for GATA-1 and the SCL complex. Furthermore, the chromatin at this site becomes activated as judged by acetylation of histone H3 and H4 (H3ac2 and H4ac4) and methylation of histone H3 (H3K4me2). Based on these data we postulate that an active transcriptional complex binding this new GATA site created by the SNP-mutation, could distract the upstream regulatory regions, which normally interact with the α globin promoter, and silence α globin gene expression. This model thus represents a new example of α globin gene down-regulation and a new mechanism by which gene expression can be perturbed during hemopoiesis.

Description: Glucocorticoids (GCs) exert powerful anti-inflammatory effects that may relate in part to their ability to restrict the differentiation and function of dendritic cells (DCs). Although these inhibitory effects are dependent upon GCs binding to nuclear glucocorticoid receptors (GRs), fine-tuning of GR signaling is achieved by prereceptor interconversion of cortisol that binds GRs with high affinity and cortisone that does not. We show for the first time that human monocyte-derived DCs are able to generate cortisol as a consequence of up-regulated expression of the enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1). Immature DCs demonstrate selective enhancement of 11β-HSD1 reductase activity, leading to increased conversion of inactive cortisone to active cortisol. Enhancement of GC bioavailability is maintained or increased upon terminal differentiation induced by signals associated with innate immune activation. In marked contrast, maturation induced by CD40 ligation leads to a sharp reduction in cortisol generation by DCs. The differentiation of DCs from monocyte precursors is inhibited at physiologic concentrations of inactive cortisone, an effect that requires activity of the 11β-HSD1 enzyme. In conclusion, prereceptor regulation of endogenous GCs appears to be an important determinant of DC function and represents a potential target for therapeutic manipulation.

Description: We have conducted a Phase II trial (TIDEL) in de-novo CML patients using imatinib 600 mg initially, increasing to 800 mg if specified response criteria were not met, including: major cytogenetic response (MCR) at 6 months; complete cytogenetic response (CCR) at 9 months, and ≥4 log reduction in BCR-ABL at 12 months as determined by Real Time Quantitative PCR (RQ-PCR). We have previously shown in this cohort that the mean daily dose (MDD) of imatinib in the first 6 months was predictive for the log reduction of BCR-ABL at 12 months. A BCR-ABL reduction of ≥3 logs from the standardized baseline (major molecular response, MMR) is highly predictive for progression free survival for de-novo patients treated with 400 mg of imatinib. The aim of the current exploratory analysis was to assess the impact of MDD and cytopenias in the first and second 6 months of imatinib therapy on the molecular response at 12 and 24 months. Out of 101 accrued patients 81 were assessable for the 24 month molecular response (median age 47 years, range 21–75). Four patients were not yet assessable and 16 patients were off study, 7 for non-disease related reasons and 9 because of treatment failure, 3 of whom progressed to blast crisis. Dose increases to 800 mg for failure to achieve response criteria as mandated by the protocol were activated in 7 patients before 12 months and in most of the remaining patients after 12 months. Dose intensity through periods of neutropenia was maintained by protocol specified use of Filgrastim. By 12 months 89%, 45% and 13% had achieved CCR, ≥3 log and ≥4 log reduction respectively. By 24 months 92%, 65% and 29% achieved these response levels. MDD 1st 6 months, 2nd 6 months No. % MMR at 12 months P value % MMR at 24 months P value LR at 24 months P value LR is mean log reduction in BCR-ABL. The P values refer to paired comparisons of the [600,600] dose with each of the other 3 dose patterns 600,600 51 56 89 3.61 400–599,600 18 50 NS 61 0.02 2.94 0.006 600, 400–599 6 50 NS 67 NS 3.15 NS 400–599,400–599 10 22 0.08 43 0.009 3.1 NS The percentage of patients achieving MMR at 24 months in the [600, 600] group (89%) was also significantly different (P=0.009) from the percentage (50%) in the [500–599, 600] group. Thus even minor dose reductions in the first 6 months appears to have a strong bearing on the achievement of MMR at 24 months. Dose modifications in the second 6 months may have less impact on molecular response. Dose modifications were commonly due to cytopenia. However, thrombocytopenia and neutropenia in the first 12 months were not significantly associated with MMR at 12 or 24 months. Sokal prognostic scores were not significantly different in patients who had reduced MDD in the first 6 months. We conclude that a more dose intense approach to the treatment of newly diagnosed CML achieves better rates of MMR than lower doses, and that maintenance of dose intensity in the first 6 months of therapy is predictive of molecular response, independent of the ability to increase dose at a later point.

Description: Imatinib is a tyrosine kinase inhibitor that suppresses the growth of bcr-abl–expressing chronic myeloid leukemia (CML) progenitor cells by blockade of the adenosine triphosphate (ATP)–binding site of the kinase domain of bcr-abl. Imatinib also inhibits the c-abl, platelet-derived growth factor (PDGF) receptor, abl-related gene (ARG) and stem-cell factor (SCF) receptor tyrosine kinases, and has been used clinically to inhibit the growth of malignant cells in patients with CML and gastrointestinal stromal tumors (GISTs). Although initially considered to have minimal effects of normal hematopoiesis, recent studies show that imatinib also inhibits the growth of some nonmalignant hematopoietic cells, including monocyte/macrophages. This inhibition could not be attributed to the known activity profile of imatinib. Here, we demonstrate for the first time that imatinib targets the macrophage colony-stimulating factor (M-CSF) receptor c-fms. Phosphorylation of c-fms was inhibited by therapeutic concentrations of imatinib, and this was not due to down-regulation in c-fms expression. Imatinib was also found to inhibit M-CSF–induced proliferation of a cytokine–dependent cell line, further supporting the hypothesis that imatinib affects the growth and development of monocyte and/or macrophages through inhibition of c-fms signaling. Importantly, these results identify an additional biologic target to those already defined for imatinib. Imatinib should now be assessed for activity in diseases where c-fms activation is implicated, including breast and ovarian cancer and inflammatory conditions.

Description: Most patients with de novo chronic myeloid leukemia (CML) achieve good responses to imatinib, but the rate and degree of molecular response is variable. We assessed the inhibitory concentration 50% for imatinib (IC50imatinib) in 62 patients with de novo chronic-phase CML as a predictor of molecular response. IC50imatinib was determined in pretherapy blood samples by measuring the in vitro imatinib-induced reduction of the phosphorylated form of the adaptor protein Crkl (CT10 regulator of kinase like). There was marked variability between patients, with IC50imatinib ranging from 0.375 to 1.8 μM (median, 0.6 μM). Patients with low IC50imatinib (IC50 ≤ 0.6 μM; n = 36) had a 36% probability of achieving 2-log reduction in BCR-ABL (breakpoint cluster region-abelson) by 3 months compared with 8% in patients with high IC50imatinib (n = 26) (P = .01). The IC50imatinib was also predictive of molecular response at 12 months, with 47% of patients in the low IC50imatinib group achieving 3-log reduction and 23% in the high IC50imatinib group (P = .03). The predictive power of IC50imatinib was particularly strong in patients with low Sokal scores. These data provide strong evidence that intrinsic sensitivity to imatinib is variable in previously untreated patients with CML, and the actual level of BCR-ABL kinase inhibition achieved is critical to imatinib response. The IC50imatinib potentially provides a new prognostic indicator for molecular response in patients treated with imatinib. (Blood. 2005; 106:2520-2526)

Description: IL-21 is a cytokine produced by activated CD4+ T cells that augments the growth, survival, and function of NK, T and phagocytic cells. IL-21 also plays an important role in regulating B cell proliferation and differentiation. In a previous study of cynomolgus monkeys treated with recombinant human IL-21, we demonstrated increased numbers of NK cells and phagocytic cells expressing Fcγ RI and III, enhancement of ex vivo antibody dependent cellular cytotoxicity (ADCC) activity, altered lymphocyte trafficking, and improved B cell depletion with subclinical doses of rituximab. The current study aimed to extend these findings to a clinically relevant rituximab dose regimen, and to evaluate the safety and tolerability of IL-21 administration in this setting. Three groups of cynomolgus monkeys (n=8) were treated by IV injection once weekly for four weeks with rituximab (10 mg/kg), concurrently with vehicle or IL-21 (0.3 or 1.5 mg/kg). Standard toxicology data were collected. The number and activation state of peripheral blood leukocyte subsets were determined by flow cytometry. Effects on B cells within spleen and lymph nodes were evaluated by immunohistochemistry. Co-administration of rituximab and IL-21 was well tolerated at both dose levels of IL-21 tested. The primary toxicological responses to the combination treatment were moderate anemia and thrombocytopenia, which were IL-21 dose dependent and similar in magnitude to those observed in previous IL-21 toxicology studies. In animals treated with rituximab alone, circulating B cells were depleted to a nadir 0.3–3 % of baseline on Day 11 of the study (Figure 1). In contrast, significantly lower B cell nadirs were observed following the first treatment with rituximab and IL-21 combination therapy (p〈 0.01, ANOVA). In animals receiving the 1.5 mg/kg dose of IL-21, circulating B cells were depleted to a nadir 0 −1 % of baseline on Day 4, followed by partial recovery and further depletion to 0.3 – 2% of baseline on Day 11. More importantly, B cell recovery was significantly delayed in the group treated with rituximab and 1.5 mg/kg IL-21, compared to rituximab treatment alone (p〈 0.05, ANOVA/PLSD). Thirty days following the fourth and final treatment, B cell numbers had recovered to only 6% of baseline, compared to 22% in the rituximab monotherapy treated group. Additionally, coadministration of IL-21 with rituximab increased circulating activated monocytes, granulocytes and NK cells compared with rituximab monotherapy. In conclusion, addition of IL-21 to a standard weekly, 4-dose regimen of IV rituximab was well-tolerated in cynomolgus monkeys and resulted in more complete, rapid and durable depletion of circulating CD20-expressing target cells. As depletion of B cells in this model serves as a surrogate for clearance of CD20-expressing malignant cells, these data support the evaluation of IL-21 and rituximab combination therapy in patients with advanced CD20 positive malignancies. Figure Figure

Description: Background and methods: The IRIS study initiated in June 2000 compared treating newly diagnosed CML-CP pts with IM or interferon+ara-C. Pts who achieved a complete cytogenetic response (CCyR) had BCR-ABL transcript numbers in the blood measured serially by real-time quantitative PCR. Results were expressed as a log reduction from a standardized baseline value for untreated pts. We showed previously that by 1 year an estimated 40% of pts who started IM at 400 mg/day had achieved a major molecular response (MMR) defined as ≥3 log reduction in BCR-ABL levels. Of the 136 pts who achieved MMR at one year, all survived at 42 months without evidence of progression to advanced phase whereas 5% of the 94 pts who failed to achieve MMR at one year had progressed. Although routine PCR sampling was interrupted January 2003, (24 months after end of study recruitment), 6-monthly monitoring was resumed in August 2004 for all pts on study regardless of CCyR status. PCR data analyzed up to February 2005 are included here. Objectives: To evaluate level of BCR-ABL transcripts after approximately 4 years on IM in pts who had transcript numbers measured in CCyR at 1 year. Of the 553 pts who started treatment with IM, this analysis is based on the 101 pts who had achieved CCyR within 1 year, who received first-line treatment at least for 24 months and who had blood collected for measuring transcript numbers at 1 year and 4 years after starting treatment. Results: At 1 year BCR-ABL transcript levels had fallen by at least 3 log in 47 (46%) of the 101 pts. At the most recent measurement at or after 44 months from start of study (median 49 months, range 44 – 55), 76 (75%) pts showed a reduction of at least 3 logs. Of these 76 pts, 39 (51%) had already had a ≥3 log reduction at year one, whereas 37 (49%) had not. Conversely 8 of the 47 pts with ≥3 log reduction at 1 year had log reductions