ALBERT

Description: Point mutations were found in the adenosine triphosphate (ATP) binding region of BCR/ABL in 12 of 18 patients with chronic myeloid leukemia (CML) or Ph-positive acute lymphoblastic leukemia (Ph+ ALL) and imatinib resistance (defined as loss of established hematologic response), but they were found in only 1 of 10 patients with CML with imatinib refractoriness (failure to achieve cytogenetic response). In 10 of 10 patients for whom samples were available, the mutation was not detected before the initiation of imatinib therapy. Three mutations (T315I, Y253H, and F317L present in 3, 1, and 1 patients, respectively) have a predicted role in abrogating imatinib binding to BCR/ABL, whereas 3 other mutations (E255K, G250E, and M351T, present in 4, 2, and 2 patients, respectively) do not. Thus we confirm a high frequency of mutations clustered within the ATP-binding region of BCR/ABL in resistant patients. Screening may allow intervention before relapse by identifying emerging mutations with defined impacts on imatinib binding. Certain mutations may respond to higher doses of imatinib, whereas other mutations may mandate switching to another therapeutic strategy.

Description: Imatinib-treated chronic myeloid leukemia (CML) patients with acquired resistance commonly have detectable BCR-ABL kinase domain mutations. It is unclear whether patients who remain sensitive to imatinib also have a significant incidence of mutations. We evaluated 144 patients treated with imatinib for BCR-ABL kinase domain mutations by direct sequencing of 40 accelerated phase (AP), 64 late chronic phase (≥ 12 months from diagnosis, late-CP), and 40 early-CP patients. Mutations were detected in 27 patients at 17 different residues, 13 (33%) of 40 in AP, 14 (22%) of 64 in late-CP, and 0 of 40 in early-CP. Acquired resistance was evident in 24 (89%) of 27 patients with mutations. Twelve (92%) of 13 patients with mutations in the adenosine triphosphate (ATP) binding loop (P-loop) died (median survival of 4.5 months after the mutation was detected). In contrast, only 3 (21%) of 14 patients with mutations outside the P-loop died (median follow-up of 11 months). As the detection of mutations was strongly associated with imatinib resistance, we analyzed features that predicted for their detection. Patients who commenced imatinib more than 4 years from diagnosis had a significantly higher incidence of mutations (18 [41%] of 44) compared with those treated within 4 years (9 [9%] of 100), P 〈 .0001. Lack of a major cytogenetic response (MCR) was also associated with a higher likelihood of detecting a mutation; 19 (38%) of 50 patients without a MCR had mutations compared with 8 (8.5%) of 94 with an MCR, P 〈 .0001. In conclusion, the detection of kinase domain mutations using a direct sequencing technique was almost always associated with imatinib resistance, and patients with mutations in the P-loop had a particularly poor prognosis. (Blood. 2003; 102:276-283)

Description: The IRIS trial demonstrated that imatinib at 400 mg/day was superior to alpha interferon as first line therapy for chronic phase CML. To assess the tolerability and efficacy of higher doses of imatinib in this setting we are conducting a Phase II trial (TIDEL) using imatinib 600 mg initially, increasing to 800 mg if specified response criteria are not met: complete hematologic response (CHR) at 3 Mo; major cytogenetic response (MCR) at 6 Mo; complete cytogenetic response (CCR) at 9 Mo, and 〉4 log reduction in BCR-ABL at 12 Mo. Filgrastim was used in cases of neutropenia to maintain dose intensity. Of 103 patients enrolled, 8 came off study in the first 12 Mo (2 unrelated deaths, 3 blast crisis, 3 poor response). 80 patients are currently assessable at 12 Mo (median age 47 years, range 21–75). Protocol mandated dose increases to 800 mg for failure to achieve MCR or CCR targets were activated in 7 patients before 12 months. We made a historical comparison of the best response by 12 Mo to responses in the IRIS trial (95% confidence intervals). Response rates at 12 Mo MCR (0–35% Ph+) CCR (0% Ph+) MMR (≥3 log reduction in BCR-ABL) 400mg - imatinib arm of IRIS n=556 84.1% (81.0 – 87.2%) 69.3% (65.3 – 73.2%) 40% (NA) 600mg - imatinib in TIDEL trial n=80 94.2% (87.3 – 97.5%) 88.5% (80.3 – 93.5%) 47.4% (37.5 – 57.6%) P-value for z-test 0.0004

Description: Pro-inflammatory monocyte-derived cytokines (monokines) such as interleukin (IL)-12, IL-15 and IL-18 work in concert to quickly induce IFN- γ in natural killer (NK) cells, while the anti-inflammatory monokine transforming growth factor beta 1 (TGF-β1) can suppress NK cell IFN- γ production. Here, we provide new molecular evidence as to how these monokines carry out their opposing functions. In the human NK-92 cell line we show that Smad proteins, mediators of TGF- β1 signaling, directly inhibit IFN- γ production. Using chromatin immunoprecipitation (ChIP), we found that both Smad2 and Smad4 associate with the IFN- γ promoter in the proximal region but not in other regions, such as exon 4. Luciferase reporter assays in 293T cells indicated that Smad2 alone, which does not possess a DNA binding domain, had little effect on the inhibition IFN-γ of promoter activity. In contrast, Smad3 and Smad4, both of which have DNA binding activity, dramatically suppressed IFN-γ promoter activity. Co-transfection of Smad2 and Smad4, or Smad3 and Smad4, in 293T cells resulted in synergistic repression. Interestingly, the Smad proteins also inhibited the transactivation of the IFN-γ promoter by T-BET, a recently discovered transcription factor controlling IFN- γ production in mouse T cells and NK cells. This was consistent with our data showing that TGF-β1 inhibited T-BET expression in human NK cells. Using IFN-γ promoter deletion constructs, we mapped the region responsible for Smad-mediated inhibition to the -204 proximal promoter, where Smad binding elements are enriched. In parallel, using micro-array analysis, western-blotting and semi-quantitative PCR in human primary NK cells and the NK-92 cell line, we demonstrated that the combination of the pro-inflammatory monkines IL-12 with IL-15 or IL-18 was able to antagonize multiple components of TGF- β1 signaling, including: 1) inhibition of the expression and the phosphorylation of Smad2; 2) suppression of Smad3 expression; and 3) down-regulation of the expression of the TGF- β receptor II subunit. Moreover, although these monokines had no effect on Smad4 expression, using ChIP assay we observed that Smad4 dissociated from the proximal IFN-γ promoter upon co-stimulation of NK cells with IL-12 and IL-18. In summary, our data show that TGF- β1 suppresses IFN- γ production via the direct binding of Smad proteins to the proximal IFN-γ promoter and via inhibition of T-BET, while the pro-inflammatory cytokines serve to inhibit or down modulate TGF- μ signaling components and reverse Smad binding to the proximal IFN-γ promoter. Thus, we suggest that NK cell production of IFN-γ is controlled by the integration of anti-inflammatory and pro-inflammatory monokine signals, leading to reciprocal antagonism.

Description: Molecular characterization of human natural killer (NK) cells will require targeted gene delivery to inhibit and activate specific signaling pathways, yet to our knowledge, an effective means to deliver such products for long-term gene expression without disrupting normal cellular processes has not been described. In this study we have developed a retroviral strategy to effectively express gene products in the NK cell, whereby its effector functions of cytotoxicity and cytokine production remain intact. Using an EBV/retroviral hybrid vector PINCO, we demonstrate infection of human peripheral blood NK cells with simultaneous expression of a marker for infection - the enhanced green fluorescent protein (EGFP) - along with various genes of interest. This technique results in successful infection of the CD56dim NK population that predominates among human peripheral blood NK and is the effector of antibody-dependent cellular cytotoxicity (ADCC) and natural killing. In addition, we demonstrate infection of the CD56bright NK subset as well as the NK-92 and NK-L cell lines. Finally, we modify PINCO to express a cytoplasmically truncated murine CD8 molecule in place of GFP. The resulting vector enables us to transduce NK cells with multiple genes of interest simultaneously and provides an alternative purification method to FACS by using magnetic beads. In summary, we have devised an efficient and highly reproducible methodology for the targeted delivery of gene products to human NK cells that should now provide opportunities to dissect the molecular processes critical to normal NK cell physiology and to genetically manipulate NK cell populations prior to their administration in cancer therapy.

Description: Interleukin 21 (IL-21) is an IL-2 family cytokine produced by activated CD4+ T cells. Potent effects of IL-21 have been observed on the growth, survival, and functional activation of T cells, B cells, and natural killer (NK) cells. A Phase I clinical trial of IL-21 in metastatic melanoma and renal cell carcinoma is currently in progress. We recently reported that IL-21 significantly enhanced rituximab mediated clearance of CD20+ lymphoma cell lines both in vitro and in vivo, and that these effects were potentially mediated through IL-21 enhancement of NK cell capacity to effect antibody dependent cellular cytotoxicity (ADCC). Specifically, NK cells treated with IL-21 showed increased cytotoxicity, granzyme B and IFNg production. Current studies aim to further evaluate the mechanisms by which IL-21 enhances ADCC. A number of observations suggest a multi-factorial basis for IL-21 synergy with rituximab. In a xenograft tumor model, SCID mice were injected IV with HS Sultan cells on day 0. Treatment with recombinant murine IL-21 (mIL-21; starting day 1) combined with rituximab (starting day 3) resulted in significantly increased survival (70% vs. 20% on day 100), compared to rituximab alone. In separate studies, the spleens of mice treated with mIL-21 showed increased numbers of activated macrophages and granulocytes. As macrophages and granulocytes can participate in ADCC, IL-21 synergy with rituximab in vivo may be partly dependent on its activation of these cell types. We have also evaluated whether direct effects of IL-21 on lymphoma cells contribute to enhancement of rituximab efficacy. The xenogeneic B lymphoma models in which IL-21 plus rituximab exhibited enhanced survival are highly aggressive and these models were not shown to respond to treatment with mIL-21 alone. In vitro studies were performed to determine if IL-21 could potentiate the growth inhibitory and pro-apoptotic effects of rituximab. In the absence of effector cells synergistic interaction was not observed. In addition, we tested the ability of IL-21 to enhance cytotoxicity when combined with antibodies targeting non-hematopoietic tumor cells (e.g. trastuzumab). Human NK cells treated with IL-21 displayed significantly increased cytotoxicity in ADCC assays using trastuzumab to target breast cancer cells expressing varying levels of HER-2 antigen. In summary, the current evidence suggests that IL-21 can enhance antibody-mediated tumor cell lysis through activation of multiple effectors of ADCC. Thus IL-21 may prove to be broadly applicable to monoclonal antibody therapy of cancer.

Description: The majority of patients with chronic phase (CP) CML treated with imatinib achieve a CCR, which is associated with a high progression-free survival (PFS). Nevertheless, BCR-ABL mutations have been observed in patients in ongoing CCR. We previously reported that virtually all patients with mutations develop evidence of resistance. The current analysis of 211 patients with CP CML aimed to determine the incidence of mutations in those who achieve CCR, assess whether the incidence varies with increasing duration of CCR, and evaluate the rate of loss of response in those who do develop mutations. BCR-ABL levels measured by quantitative RT-PCR at 1–6 month intervals were used to guide the frequency of mutation analysis. All patients were screened for mutations at least 6 monthly. Of the 211 patients, 159 (75%) achieved CCR after a median of 3 months (25th to 75th percentile 3–6 months), and were then followed for a median of 12 months after achieving CCR (25th to 75th percentile 6–28 months). Twelve patients lost CCR and this was associated with the development of a mutation in 8 (67%). The median time to loss of CCR was 3 months after the mutation was detected (range 1–10 months). Three of these 12 patients proceeded to transplant, and 9 received an increased imatinib dose (4 maintaining a major cytogenetic response (MCR); 3 re-establishing CCR). Only 1 of the 12 lost a complete hematologic response (CHR) (patient had 2 mutations) and none have progressed to blast crisis (BC) with a median follow-up of 9 months (range 6–38 months) including 3 patients with P-loop mutations, which have been associated with a poorer prognosis. As well as the 8 patients who lost CCR after a mutation was detected, mutations were detected in 3 patients who have maintained CCR; 2 had an increased imatinib dose. The detection of a mutation was associated with a rise in the BCR-ABL level in all patients with mutations. Of the 159 patients in CCR, mutations were detected in 11 of 25 (44%) with a 〉2-fold rise in BCR-ABL whereas none of 131 patients with stable or decreasing BCR-ABL had a mutation detected (P

Description: Imatinib therapy was rapidly introduced for CML because of its efficacy and tolerability, so ongoing scrutiny is warranted to elucidate any effects not seen in registration trials. Imatinib strongly inhibits several tyrosine kinases (abl, arg, kit, and pdgfr’s) with functions in normal tissues. Mice lacking these genes have impaired male fertility (abl or kit knockouts), B- and T- cell lymphopenia (abl), emphysema (pdgf) and glomerular dysfunction (pdgf). Hence, we prospectively analysed chronic-phase CML patients on higher-dose imatinib (Hughes TP et al. Abstract #95 ASH 2003) for fertility, immunoglobulin levels, lymphocyte sub-sets and pulmonary function over 1 year. 39/103 patients participated in the safety sub-study, with median age 50 (range 28–75), on median daily imatinib doses of 558–575mg. In 20 males tested, median serum testosterone (in nmol/L) changed little, from 12.8 at baseline to 14.7 at 6 months, to 11.4 at 1 year (Wilcoxon p=0.05). Serum LH, FSH and oestradiol were unchanged. Only 2 patients had repeat sperm counts: one recovered from azoospermia to have low counts; the other had normal counts at baseline and 6 months. In 20 females, there were no changes in median serum progesterone, oestradiol or LH. FSH (in U/L) fell from 25 to 5 at 6 months, but rose to 21 at 1 year (p=0.03). Median serum IgG levels (in g/L; n=34) fell from 10.5 to 9.8 at 6 months, to 8.6 at 1 year (p

Description: The ability to predict which CML patients respond poorly to imatinib may facilitate a more aggressive approach to therapy in these patients. We have used western blotting to assess the phosphotyrosine content of the adaptor protein Crkl (p-Crkl) -a major substrate for BCR-ABL. PB was collected from 57 newly diagnosed CML patients prior to the initiation of therapy and dose dependent inhibition of Crkl phosphorylation, in the presence of imatinib, was measured following incubation of primary cells for 2 hours with varying doses of imatinib. Using this assay we were able to determine that the median IC50 for imatinib was 0.6 uM (R 0.375–1.8uM). For statistical analysis IC50’s were then grouped into low and high relative to the median, and compared to molecular response achieved at both 3 and 12 months. Using Log Rank Survival Analysis there was a significant difference in the probability of achieving a 2 log depletion of BCR-ABL (measured by quantitative real-time Q-PCR) by 3 months between the two groups (Low IC50 30% n=31, High IC50 4% n=26; p=0.023 n=57). In addition the probability of achieving a 3 log reduction of BCR-ABL at 12 months (MMR, major molecular response) was significantly different (Low IC50 47%, High IC50 23%, p=0.034 ). We therefore assessed whether the IC50 was a surrogate measure of the Sokal Score or was an independent predictor of response. When we examined the median Sokal scores of both the low and high IC50 groups we found that there was no significant difference between the two groups (Low IC50 = 1.0, High IC50 = 0.9 p=〉0.05 ) suggesting that IC50 was not a surrogate measure of Sokal. Log Rank survival analysis revealed that while Sokal Score did not predict for a 2 log depletion at 3 months (Low (43% n=32) vs Intermediate (21%n=25) vs High (21% n=29) p〉0.05 ) it was predictive of a greater probability of MMR at 12 months (Low 53% vs Intermediate 42% vs High 21% p=0.044 ). The combined effect of these parameters was assessed: Probability of achieving a Major Molecular Response. All Low Sokal Intermediate Sokal High Sokal Note: Not all Sokal Scores available at the time of analysis. Low IC50 47% n=30 67% n=9 50% n=6 17% n=12 High IC50 23% n=23 20% n=10 22% n=9 0% n=4 p-value 0.034 0.037 〉0.05 〉0.05 Evidence presented here suggests that the IC50 level measured in blood cells from CML patients prior to imatinib therapy provides a statistically significant indicator of response at both early and late timepoints. Further to this, the combined measures of Sokal Score and IC50 provide a powerful tool for prediction of molecular response, which clearly delineates those patients groups who have a low probability of achieving a major molecular response. Preliminary investigations into the components of both Sokal and IC50 which may be contributing to these findings have highlighted a group of patients (n=7) who have a high blast percentage (5 to 21%) and low IC50. Unlike the majority of patients with low IC50 these patients all fail to achieve MMR. When these patients are removed from the low IC50 group the probability of MMR in the remaining patients is 71%. Further investigations into underlying mechansims for the inter-patient variation in IC50 are currently being undertaken.

Description: Malignant B-cells from a high proportion of chronic lymphocytic leukaemia (B-CLL) patients over express the multidrug resistance (MDR) -1 gene encoded transmembrane efflux pump P-glycoprotein (P-gp). Inhibition of glucosylceramide synthesis has been shown to correlate with the expression and function of P-gp and sensitise cells to cytotoxic agents. We analysed the ability of glucosylceramide synthetase (GCS) inhibitors N-butyl-deoxygalactonojirimycin (OGB-1, 500μM) and N-nonyl-deoxygalactonojirimycin (OGB-2, 100μM) to sensitise B-CLL cells to conventional cytotoxic drugs 2-chlorodeoxyadenosine (CdA), chlorambucil (Chl) and fludarabine (FdR) using the in vitro cytotoxicity MTT assay. The effect on P-gp activity was also analysed using the calcein-AM accumulation assay and the results expressed as multidrug activity factor (MAF), where a MAF of 〉10 in the presence of a P-gp inhibitor denotes P-gp functional activity. GCS inhibitors were cultured with B-CLL cells for 24-48h before the assays were performed. The P-gp negative cell line CEM-CCRF had no MAF activity with an IC50 for vincristine (a known P-gp substrate) of 10ug/ml to 55.5ng/ml in the presence of OGB-2. In peripheral blood mononuclear cells from 3 normal volunteers (all P-gp +ve), the mean MAF value for Z.3HCL was 23.86 and for OGB-2 was 16.2. In 9/13 B-CLL samples there was P-gp functional activity in the presence of Z.3HCL with a mean MAF value of 22.15 (range 11.27–37.3). P-gp was over expressed in10/13 B-CLL samples. However, when available samples from this cohort were assessed with OGB-1 (n=4) and OGB-2 (n=13) the MAF value was