ALBERT

Description: Imatinib inhibits tyrosine kinases important in osteoclast (c-Fms) and osteoblast (platelet-derived growth factor receptor [PDGF-R], c-Abl) function, suggesting that long-term therapy may alter bone homeostasis. To investigate this question, we measured the trabecular bone volume (TBV) in iliac crest bone biopsies taken from chronic myeloid leukemia (CML) patients at diagnosis and again after 2 to 4 years of imatinib therapy. Half the patients (8 of 17) showed a substantive increase in TBV (〉 2-fold), after imatinib therapy, with the TBV in the posttreatment biopsy typically surpassing the normal upper limit for the patient's age group. Imatinib-treated patients exhibited reduced serum calcium and phosphate levels with hypophosphatemia evident in 53% (9 of 17) of patients. In vitro, imatinib suppressed osteoblast proliferation and stimulated osteogenic gene expression and mineralized-matrix production by inhibiting PDGF receptor function. In PDGF-stimulated cultures, imatinib dose-dependently inhibited activation of Akt and Crk-L. Using pharmacologic inhibitors, inhibition of PI3-kinase/Akt activation promoted mineral formation, suggesting a possible molecular mechanism for the imatinib-mediated increase in TBV in vivo. Further investigation is required to determine whether the increase in TBV associated with imatinib therapy may represent a novel therapeutic avenue for the treatment of diseases that are characterized by generalized bone loss.

Description: Pathological angiogenesis associated with wound healing often occurs subsequent to an inflammatory response that includes the secretion of cytokines such as tumor necrosis factor (TNF). Controversy exists on the angiogenic actions of TNF, with it being generally proangiogenic in vivo, but antiangiogenic in vitro. We find that whereas continuous administration of TNF in vitro or in vivo inhibits angiogenic sprouting, a 2- to 3-day pulse stimulates angiogenesis by inducing an endothelial “tip cell” phenotype. TNF induces the known tip cell genes platelet-derived growth factor B (PDGFB) and vascular endothelial cell growth factor receptor-2 (VEGFR2), while at the same time blocking signaling through VEGFR2, thus delaying the VEGF-driven angiogenic response. Notch signaling regulates tip cell function, and we find that TNF also induces the notch ligand jagged-1, through an NFκB-dependent mechanism. Enrichment of jagged-1 in tip cells was confirmed by immunofluorescent staining as well as by laser capture microdissection/quantitative reverse-transcription–polymerase chain reaction (qRT-PCR) of tip cells sprouting in vitro. Thus, in angiogenesis, the temporal expression of TNF is critical: it delays angiogenesis initially by blocking signaling through VEGFR2, but in addition by inducing a tip cell phenotype through an NFκB-dependent pathway, it concomitantly primes endothelial cells (ECs) for sprouting once the initial inflammatory wave has passed.

Description: While transplantation of unrelated umbilical cord blood (UCB) halts the progression of neurological damage in children with lysosomal storage diseases (LSD), it does not necessarily reverse the damage sustained before transplant. Isolation and expansion of central nervous system progenitor cells from UCB has potential therapeutic application in treating these patients. We have recently described the reliable isolation and expansion of oligodendrocyte-like precursor cells (OPC) from freshly collected, non-crypreserved UCB (Tracy et al. Cytotherapy. 2008, 3:1–8). We anticipate utilizing these cells as adjuvant, targeted therapy to reduce the time to donor cell correction/prevention of disease-induced CNS injury. This approach will require use of cyropreserved donor UCB. We now demonstrate the feasibility of isolating and expanding OPCs from a series of thawed, cryopreserved UCB units and comparing those cells to OPCs derived from fresh UCB units. Cryopreserved UCB units were thawed using a standard protocol employed in clinical transplantation. Mononuclear cells were isolated by either ficoll density separation or centrifugation after a dextran-albumin wash. Fresh UCB units underwent hetastarch depletion of red blood cells then mononuclear cell isolation by ficoll density gradient separation. Both types of cells were plated at 3×10(6) cells/ml in media containing platelet derived growth factor, neurotopin 3, vascular endothelial growth factor, and triiodothyronine. All UCB unit cultures were trypsinized at 21 days, counted, then characterize by flow cytometry after being fixed, permeablized, and labeled with the following antibodies: anti-oligidendrocyte marker 4 (O4), anti-oligidendrocyte marker 1 (O1), anti-myelin basic protein (MBP). To examine phenotypic changes over time, cultures from two units (one thawed, one fresh) were also analyzed weekly over 4 weeks. On flow cytometric analysis, 72% of thawed UCB units yielded O4-expressing cells as at least 20% of total events compared with 94% of fresh UCB units (Table 1). Average oligodendrocyte yield per UCB unit was less for thawed units (9.65×10(5)) compared with fresh (3.77×10(6)). However, within the gated population, expression of O1, O4, and MBP was similar between thawed and fresh cords. We also noted early expression of the preoligodendrocyte marker O4 by 1–2 weeks in culture, followed by increasing expression of mature oligodendrocyte markers O1 and MBP over the subsequent 2–4 weeks (Table 2). Our results demonstrate that oligodendrocyte precursor cells can be derived reliably from thawed, cryopreserved UCB units, and suggest the feasibility of using these cells in human clinical trials. Table 1: Characteristics of UCB-Derived Oligodendrocytes Thawed UCB Units Fresh UCB Units Total Units Cultured 18 16 Mean # mononuclear cells plated/UCB Unit 2.55×10(8) 1.4×10(8) Final oligodendrocyte cell count/UCB Unit 8.41×10(5) 3.7×10(6) Units with O4 expression 〉20% of total events 13 (72%) 15 (94%) Events in gate as % total events 58.26 77.83 O4 expression (% of gated) 83.66 86.45 O1 expression (% of gated) 88.13 86.29 MBP expression (% of gated) 31.10 31.41 Co-expression of MBP-O1 (% of gated) 28.50 31.86 Table 2: Expression of Markers by UCB-Derived Oligodendrocytes Thawed UCB Unit Fresh UCB Unit Day O1 MBP MBP+ O1 Day O1 MBP MBP+ O1 Day 7 95.75 10.14 9.45 Day 7 68.78 25.13 12.11 Day 14 98.07 35.93 35.74 Day 14 97.71 34.03 33.75 Day 21 95.24 35.43 31.80 Day 21 99.22 34.82 34.72 Day 28 99.69 51.08 51.04 Day 28 96.49 24.10 23.40

Description: Developmental intermediates of human natural killer (NK) cells are found within secondary lymphoid tissue (SLT), and five distinct stages of these intermediates have been identified. While it is well documented that developing NK cells are reliant on interleukin (IL)-15 as a survival factor, it is likely that additional cytokines and growth factors are required for complete NK cell differentiation. Microarray transcriptional profiling of purified stage 1–4 cells from human tonsil and stage 4 and 5 cells from peripheral blood (PB) identified a developmental window of interleukin-1 receptor 1 (IL-1R1) messenger RNA (mRNA) expression restricted to stages 2 and 3. We confirmed this finding by quantitative RT-PCR, and analysis of IL-1R1 surface protein expression revealed that, on average, 81% of stage 3 immature NK cells are IL-1R1(+), whereas the majority of cells from stages 1, 2, and 4 are IL-1R1(−). When cultured in vitro with IL-1β, a physiologic ligand for IL-1R1, cells from all four stages died within 48 hours, consistent with an absolute requirement for IL-15 as a survival factor. However, the combination of IL-1β and IL-15 led to a significant and reproducible 4.64±−0.68–fold increase in stage 3 cell number over that seen with IL-15 alone (p 〈 0.0005). This phenomenon was completely restricted to stage 3 immature NK cells, and is attributed to increased proliferation. The effects of IL-1β were abrogated by a molar excess of IL-1 receptor antagonist (IL-1RA), a physiologic competitor for IL-1R1 binding. Collectively, our data indicate that IL-1R1 expression fluctuates dramatically during NK cell development, and that unique responses of IL-1R1(+) stage 3 cells to IL-1β and IL-15 govern the expansion of these immature NK cells. Our findings support a model in which IL-1β promotes stage 3 proliferation and survival in vivo, driving stage 3 cells to be the most prevalent NK cell intermediates within SLT.

Description: Expression of BCR-ABL1 is the hallmark of chronic myelogenous leukemia (CML) and a subset of de novo acute lymphoblastic leukemia (ALL), but the factors determining disease lineage, and progression of CML to myeloid or lymphoid blast crisis, are incompletely understood. We recently reported deletion of IKZF1 (encoding the lymphoid transcription factor Ikaros) in 85% of de novo pediatric and adult BCR-ABL1 ALL, and in lymphoid blast crisis in a small cohort of CML cases (Nature2008;453:110), suggesting that IKZF1 deletion is important in the pathogenesis of BCR-ABL1 lymphoid leukemia. To identify genetic determinants of disease stage and blast crisis lineage in CML, we have now performed high-resolution, genome wide analysis of DNA copy number abnormalities (CNA) and loss-of heterozygosity (LOH) and candidate gene resequencing in a cohort of 90 CML patients that included 64 samples obtained at chronic phase (CP), 15 samples at accelerated phase (AP), 9 lymphoid blast crisis (LBC) and 22 myeloid blast crisis (MBC) samples. Importantly, 25 patients had sequential samples (CP and/or AP, as well as blast crisis samples) enabling analysis of lesions acquired at progression to blast crisis. All blast crisis samples were flow sorted to at least 90% purity prior to DNA extraction. Germline samples for 28 cases obtained at remission or by flow sorting of blast crisis samples were also examined. Affymetrix SNP 6.0 arrays, interrogating over 1.87 million genomic loci, were used for 85 samples, and 500K arrays for the remainder. Identification of tumor-specific (somatic) copy number analysis was performed by directly comparing CML samples to matched germline samples were available, or by filtering results against databases of inherited copy number variants for samples lacking germline material. Genomic resequencing of IKZF1, PAX5 and TP53 was performed for all AP, LBC and MBC samples. There were few CNAs in CP-CML (mean 0.27 deletions and 0.07 gains per case), with no recurring lesions identified apart from deletions or gains at the chromosomal breakpoints of BCR and ABL1 (3 cases each). Notably, the size of these translocation associated deletions was highly variable, ranging from 6kb (one ABL1 deletion) and 15 kb (one BCR deletion) to deletions extending to the telomeres of chromosomes 9 and 22. No significant increase in lesion frequency was identified in AP cases (0.14 deletions and 0.9 gains per case), however the number and cumulative extent of genomic aberrations was significantly higher in both lymphoid and myeloid blast crisis samples. LBC cases had a mean of 8.1 deletions/case (P

Description: We have previously demonstrated significant interpatient variability in the IC50imatinib, a measure of the intrinsic sensitivity of a patient to imatinib induced kinase inhibition. Furthermore, this measure is predictive of the achievement of major molecular response (MMR 〉 3 log reduction in BCR-ABL) in de-novo CML patients treated with imatinib (n=60)1. In an expanded patient pool (n=116) we now perform an evaluation of the IC50 as a predictor of response, and address the IC50imatinib as a guide to dose selection. Samples were obtained with informed consent from de novo CML patients enrolled to either the TIDEL (600mg imatinib) or TOPS (randomised 400mg vs 800mg imatinib) trials. Blood was collected pre therapy, and the IC50 was performed as previously1. Outcome data was assessed using Kaplan Meier Analysis and the log rank test was used to assess statistical significance. In our previous analysis the IC50imatinib was divided about the median value for the cohort (0.6μM) into low and high IC50, with a significantly greater proportion of patients with low IC50imatinib achieving MMR by 12 months. In this expanded patient pool, we confirm this finding (0.95uM achieve MMR on 400mg, and that this is statistically significantly when compared to all other groups. At 600mg while there is no overall significant difference there is a statistically relevant difference between groups 1, 2 and 4 as indicated. In contrast, at 800 mg the effect of IC50imatinib is overcome. MMR by 12 months Total 400mg 600mg 800mg p value Group1 0.50.70.95μM 32% (28) 0% (7)* 22% (9)* 58% (12) 0.016 P value 0.042 0.018 0.108 0.778 Table 1: Dividing the patients into quartile based on the IC50 imatinib and assessing the Impact of dose on the achievement of MMR by 12 month. *p value 0.7μM fail to achieve a 2 log reduction when treated with 400mg (IC50 0.7μM 33% p=0.034), and 600mg (IC50 0.7μM 22% p=0.036). However, there is no significant difference in the 800mg patient cohort (IC50 0.7μM 14% p=0.79). This analysis confirms that the IC50imatinib, is predictive of imatinib response. Patients with an IC50imatinib 0.7μM) may benefit from higher dosing regimens (p=0.012). Thus, the accurate assessment of IC50imatinib could support dose optimization strategy for patients with a suboptimal response.

Description: Molecular analysis is recommended for monitoring patients (pts) with CML. For imatinib treated pts in chronic phase (CP), molecular analysis provides important prognostic information. A major molecular response (MMR, BCR-ABL ≤0.1% IS (international scale)) is associated with favourable progression free survival and is a primary endpoint of clinical trials. The 3 month (m) BCR-ABL level is predictive of MMR and almost all de-novo pts with values ≤1.0% IS subsequently achieve MMR. The second generation tyrosine kinase inhibitors nilotinib and dasatinib (2TKI) have demonstrated efficacy for CP pts who fail imatinib therapy due to resistance or intolerance. However, treatment failure associated with the presence of a limited spectrum of resistant mutations is evident. Furthermore, it has recently been suggested that failure to achieve a major cytogenetic response (MCR) by 12m defines inadequate response and these pts should be considered for alternative therapies (Cortes et al, Blood,2008,112,516). Pts in minor cytogenetic response or complete hematologic response at 12m had a projected 1 year progression rate of 17% compared to 3% for those with MCR at 12m. The value of molecular monitoring in the setting of 2TKI has not been defined in terms of the early prediction of response or emergence of resistant mutations. We monitored BCR-ABL levels and mutation status in 155 CP pts treated with nilotinib (n=73; 400mg BD) or dasatinib (n=82; ≥100mg (76/82 70mg BD)) after imatinib failure for a median of 18m (range (r) 3–36). The BCR-ABL level at 3m of 2TKI was highly predictive of subsequent MMR, P10% IS rather than upon a significant rise. The rise associated with emergent mutations when BCR-ABL was ≤10% IS was significantly higher: median 7.3-fold, P10% IS are at risk of acquiring 2TKI resistant mutations and would benefit from regular mutation screening until BCR-ABL falls below 10% IS. Thereafter, a significant rise of 〉5-fold in BCR-ABL should trigger mutation screening. Figure Figure

Description: Aim: To develop and evaluate a DNA-based method for monitoring of MRD in CML Background: At present, monitoring of MRD in CML uses RNA and reverse transcription (RT-PCR). This leads to a number of disadvantages, including the potential for RNA degradation, requirement for reverse transcription, difficulty in standardisation and only an indirect relationship between assay result and cell number. Methods: A highly multiplexed, short-range PCR using six BCR primers and a pool of 282 ABL primers was used to amplify across the BCR-ABL translocation breakpoint and was followed by 2–3 rounds of “bottleneck PCR”, a technique recently-developed in our laboratory, which adjusts primer concentration so as to minimise non-specific amplification and facilitate highly multiplexed PCRs. The breakpoint was isolated and sequenced, patient-specific primers were synthesised, and MRD was quantified using 10 μg of DNA and a 3 round nested quantitative PCR incorporating a Taqman probe in the third round. Samples from patients on treatment were divided and assayed both by this technique and by RT-PCR. 10 μg of DNA from a normal individual was used in each assay as a control for non-specificity. Results: The BCR-ABL breakpoint was successfully isolated and sequenced in 28 of the 29 patients studied and MRD was assayed in 38 samples from 24 patients. Follow-up samples from 4 patients were unavailable MRD was detected and measured by both methods in 22 samples, detected and measured only by DNA-PCR in 10 samples and not detected by either method in 6 samples. The limit of detection of RT-PCR was in accord with previous results which indicated it to be a mean decrease of 4.5-logs below baseline. The mean limit of detection of DNA-PCR was an MRD of 7.2 x 10−7. Assay precision was determined by performing independent replicate assays on different days and by different individuals on the 32 samples with MRD detectable by DNA-PCR. The median SD of a single assay was 0.15 log units with the range being 0.00 – 0.64 log units. Figure Figure Conclusions Monitoring of MRD by DNA-PCR is feasible in the great majority of patients with CML. In terms of possible clinical benefits, DNA-PCR is more sensitive than RT-PCR, and provides a direct measure of leukemic cell number in the individual patient. Use of DNA rather than RNA simplifies specimen collection and transport. In terms of laboratory benefits, DNA-PCR obviates reverse transcription and inter-laboratory standardisation. The principal disadvantage of the method is the initial cost, although this may be able to be amortised over several assays if monitoring is ongoing.

Description: BACKGROUND: Cytogenetic assessment to measure CML response to Glivec or Tasigna is expressed as a ratio of Ph+ CML to total cells. Molecular response determination for CML by RQ-PCR achieves a similar goal by obtaining a ratio of transcript levels of BCR-ABL expressed exclusively in Ph+ CML cells to the BCR or ABL control gene which is expressed in both Ph+ and normal cells. However when a RQ-PCR assay result is obtained, a different RQ-PCR ratio will be obtained for the same sample using BCR vs. ABL as control gene due to the intrinsic expression level differences in the two control genes. Experiments have demonstrated empirically that international standardization of RQ-PCR data obtained by assays using these different control genes is possible, thus allowing direct comparison of data. We have developed a mathematical simulation that explains the underlying basis for international standardization and this was experimentally confirmed to show how standardization is feasible in practice as a part of the Tyrosine Kinase Optimization and Selectivity (TOPS) trial. METHODS: We have mathematically simulated a correlation between theoretical cytogenetic response (Ph+ to total cell ratio) with the theoretical molecular response (BCR-ABL to control gene transcript ratio) by using the following assumptions; 1) BCR and BCR-ABL expression occur from similar promoters and likely have similar expression levels. 2) Ph+ cells have one normal BCR resulting in 1x level of BCR transcription and normal cells have two normal copies of BCR resulting in 2x level of BCR transcription (x=BCR transcription rate from one promoter). This simulation was extended to the ABL control gene as data of the relative expression level of ABL vs. BCR was available from samples in the TOPS trial, and the ABL transcript level is similar in both Ph+ and normal cells as the ABL PCR primers amplify both ABL and BCR-ABL transcripts. To experimentally test the simulation, RQ-PCR samples from patients in TOPS were exchanged between the study’s regional RQ-PCR laboratories. Each sample was assayed at 2 labs, one using BCR and the other ABL as the control gene. RESULTS: Mathematical simulation suggests that RQ-PCR ratios with BCR but not ABL as control gene has a nonlinear relationship with the Ph+ to the total cell ratio across the entire range from 0 to 100%. However at clinically relevant RQ-PCR ratio of ≤10%, irrespective of whether BCR or ABL is used as a control gene, RQ-PCR ratios are linear but with different slopes due to higher ratios obtained with ABL as control gene. Despite the different slopes, RQ-PCR ratios obtained with either control gene can be easily inter-converted via a multiplicative factor to neutralize the bias between methods as is currently recommended in international standardization. Experimental testing in the TOPS trial showed that; 1) ABL transcript copy number is significantly lower than BCR transcript copy number leading to higher RQ-PCR ratios with ABL as control gene. 2) Mathematical correction of bias between BCR and ABL as control gene allows for direct comparison of RQ-PCR ratios with an 88.2% concordance when ratios are below 10%. CONCLUSIONS: Mathematical simulation shows that for BCR and ABL as the control gene, the basis for international standardization is a consistent linear relationship that can be easily inter-converted when PCR ratios are ≤ 10%. This can be experimentally demonstrated by the high level of concordance obtained when the same sample is analyzed using different control genes. For RQ-PCR ratios 〉 10%, standardization is not possible using the currently applied neutralization of bias method of conversion, but may be possible by using a formula that accounts for the non-linear relationship of BCR as a control gene. Simulation studies also highlight the importance of not only obtaining precise but also accurate RQ-PCR ratios thus enabling consistent standardization.