bluuebunny/crossref_metadata_2025 huggingface dataset:
Combined aCGH and Mutational Analysis of CLL Patients with 17p Deletion.
Authors: Rudenko Hannah C. , Brito-Babapulle Vasantha , Gonzalez David , Martinez Pilar , Leone Paola E. , Jones Chris , Dexter Tim , Fenwick Kerry , Mackay Alan , Ashworth Alan , Matutes Estella , Catovsky Daniel , Dearden Claire , Morgan Gareth J.
Published: October 2019

We have utilised aCGH (Breakthrough Breast Cancer Research Centre 5.8K array) and mutational analysis of the TP53 gene (exons 4–10) on 74 cases of CLL to define the extent of deletion at 17p, TP53 mutational status, additional genomic changes, and how this affects clinical outcome. 17p- cases were selected by FISH (n=37, Vysis LSI P53 probe) and 37 cases were selected as being representative of the survival curve of CLL patients without 17p-. FISH identified 22 cases with TP53- in ≥ 50% of cells, 4 cases with TP53- 20–50% and 11 cases with TP53- ≤ 20%. aCGH can detect abnormalities present in >50% of cells and all of the TP53- cases greater than 50% by FISH were detected with deletion ranging between 6-20Mb in length, the majority encompassing the entire p-arm. In addition aCGH detected deletion of 17p in 5/15 cases with TP53- <50%, and 2/37 with no detectable TP53- by FISH, these deletions clustered at 17p13.3 and 17p11.2, and did not involve the TP53 gene. Cases with 17p- >20% have a poor clinical outcome (median survival 11 months, median progression free survival 3 months), the majority of these (85%) also have a mutation of TP53 whereas in cases with ≤ 20% 17p- only 10% were mutated.

The 17p- group was characterised by additional recurrent deletions involving 18p, 20p and 22q, which tended to occur as single additional events. Understanding the order in which these events occur is important, 18p- was found in 6 cases, 2 of which had <50% 17p- by FISH, suggesting that 18p- is present in a higher percentage of cells and by implication occurs prior to the 17p deletion, a similar finding was also present for the 20p- cases. 18p deletion varied in length between 5.9Mb and 12Mb, with the minimally deleted region (MDR) involving a 2.5Mb region spanning 18p11.23-p11.22. 20p- was found in 8 cases of which 3 covered almost the entire p-arm, and 5 formed 2 clusters at each end of the p-arm. 22q- was found in 8 cases, with only 1 outside of the 17p- group. Out of these 8 cases with deletion, 6 covered almost the complete q-arm but a MDR was difficult to define, however if the non-17p- case is excluded the MDR covers 1.4Mb at 22q12.3.

Recurrent abnormalities were also found on other chromosomes, but did not differ between the two groups. These included regions with previously identified abnormalities; trisomy 12 (n=11), loss of 6q14.1–24.3 (n=11), loss of 11q12.1–25 (n=17) and loss of 13q12.1–21.1 (n=6) as well as detection of novel abnormalities; gain of 4p16.3–16.1 (n=23), gain of 11p15.5–15.3 (n=22), gain of 22q11.21–13.33 (n=22) and deletions on chromosome 9 (n=9).

These results show that deletion of TP53 and mutation of the other allele are critical adverse prognostic factors. We have also defined a genetic background (18p-, 20p- and 22q-) on which these changes arise.
High Density Oligonucleotide Array CGH Analysis of CLL Reveals Areas of Recurrent Genomic Gain or Loss.
Authors: Schop Roelandt F.J. , Mancini Catherine , Chng Wee-Joo , Tschumper Renee C. , Jelinek Diane F. , Kay Neil E. , Bergsagel Peter Leif , Carpten John D. , Fonseca Rafael
Published: October 2019

The study of genomic aberrations in chronic lymphocytic leukemia (CLL) has yielded critical information, now routinely used for the prognostication of patients. An unbiased search for genome wide areas of copy number change is needed to identify possible primary genetic events in CLL and to elucidate their relationship to other known cytogenetic factors, such as deletions of 13q14, 11q23 and 17p13 and trisomy 12. Here we report on a study where we used a 60-mer oligo array-based Comparative Genomic Hybridization (aCGH, 44B platform from Agilent Technologies, Palo Alto, CA). We observed the most common genetic aberrations in CLL and in similar frequencies as would be predicted for CLL (e.g. 44% showing deletions of chromosome 13q14). Among those patients, 45% had no apparent other aberration. Of great interest, small bi-allelic deletions of chromosome 13, which predominantly involved the DLEU1 and DLEU2 locus, were detected in 4 patients (16%). Deletions of 11q23 were observed in 6 cases (24%), and while some involved the ATM locus one did not, casting doubt on the precise gene deregulated by these deletions. Large deletions of the whole 17p-arm were detected in three cases (12%). Trisomy 12 was present in only two cases (8%). No other trisomies were detected and also small regions of copy gain were uncommon. Monosomy of chromosomes 19, X and Y were present in three separate cases. Large deletions of chromosome 2p were seen in two cases, but an additional case had a small deletion involving the ALK gene. This case also had a breakpoint at chromosome 5 involving the NMP1 locus, suggestive of a t(2;5). A recent study (Mayr et al., Blood 2006) indicates that chromosomal translocations in CLL are present in 33% and were associated with a dismal prognosis. In our analysis we detected several breakpoints indicative of translocations. Detailed study is necessary to elucidate possible molecular events associated with these translocations. Other small regions of DNA copy loss were found at several locations throughout the genome. Regions of interest are shown in the table. In summary we show that multiple areas of recurrent genomic gain and loss can be identified in clonal cells of CLL and should lead to further insights into the understanding of disease pathogenesis.

Chromosome Location Gene(s) of interest Loss 6q24.3 LATS Loss 10q23.3 LG11 Loss 10q24.2 PAX2, WNT8B, TLX1, POLL, NFKB2 Loss 11p15.4 PPFIBP2 Loss 12p13 TNFRSF1A and 7 Loss 15q22.1 TCF12 Loss 19p13.3 BAX, RUVBL2 Loss 20q11.2 TP53INP2 Loss 20q13.1 WFDC13
Phase III Trial of 2-Weekly CHOP with Rituximab for Aggressive B-Cell Non-Hodgkin’s Lymphoma in Elderly Patients.
Authors: Sonneveld Pieter , van Putten Wim , Biesma Douwe , Holte Harald , van Marwijk Kooij Marinus , Kramer Mark Henri H. , Wijermans P.W. , Steijaert Monique , van Imhoff Gustaaf W. , Lugtenburg P.J. , Huijgens P.C. , Eriksson Mikael
Published: October 2019

Treatment results in elderly patients (> 65 yrs) with aggressive Non-Hodgkin lymphoma (NHL) are associated with a lower complete response (CR) rate and a worse survival (OS). Efforts have been made to improve the outcome by adding Rituximab to standard CHOP chemotherapy or by intensifying CHOP from 21 days to 14 days intervals (2-weekly CHOP, CHOP14). The Dutch HOVON and the Scandinavian Nordic groups have performed a prospective, multi-center, randomized phase III trial to compare 8 cycles of CHOP14 chemotherapy with or without 6 administrations of Rituximab (R-CHOP14), supported by G-CSF in previously untreated, older patients with intermediate or high-risk B-cell NHL. Inclusion criteria were mantle cell lymphoma, follicular lymphoma grade III or diffuse large B-cell lymphoma; intermediate or high age adjusted IPI score; CD20-positive NHL; age greater than 65 yrs. The target number was 400 patients to be accrued in 5 years based on an expected increase in failure free survival with hazard ratio HR=0.70. Based on a planned interim analysis the Data and Safety Monitoring Board stopped the trial because of a significant difference for primary endpoints. At final analysis 243 of 261 patients were eligible and evaluable. The median follow up was 20 months (range 3–46). The median age was 72 years (range 65–85 years), 80 % had DLBCL, 60 % had intermediate-high/high age-adjusted IPI score, 65 % had increased serum LDH. 64% of patients completed the planned treatment, while 22% went off treatment because of toxicity, of which 2 % was cardiovascular. 14% of patients experienced CTC grade 3–4 infections.

The number of patients who went off treatment because of toxicity was 15 % in those aged < 70 and increased with age up to 38 % in those over 75 yrs. After 3–4 cycles 16 % of the patients in the R-CHOP arm achieved a CR(u) as compared to 12 % with CHOP14; after 8 cycles the difference was 66 % vs 46 %. Overall response (PR+CRu) was 92 % and 83 %, respectively. Overall survival (OS) by Cox regression analysis adjusted for aaIPI and diagnosis was better with R-CHOP14 than with CHOP14 (relative hazard ratio HR=0.69, 95 % CI 0.46-1.05, p=0.09). The primary endpoint Failure free survival (FFS) was better in the R-CHOP14 arm (38 failures: 9 non-responders, 29 relapses, FFS2yr =55%)) than in the CHOP14 arm (65 failures: 20 non-responders, 45 relapses, FFS2yr =33%) with hazard ratio HR=0.60 (P=0.007). In the subgroup of DLBCL, FFS2yr was 62 %, while log-rank OS (p=0.05) and FFS (p=0.004) were both superior with R-CHOP14. These data indicate that in an elderly population (median age 72 yrs) with highly aggressive NHL, the CHOP14 regimen is tolerable and achievable in > 60 % of patients, and that the addition of Rituximab improves CR rate, OS and FFS.
Methylprednisolone Influences Zap-70 in B-CLL In Vitro.
Authors: Boelens Jerina , Lust Sofie , Janssens Ann , Philippé Jan , Poelman Martine , Offner Fritz
Published: October 2019

Zap-70 is accepted as a surrogate marker for mutational status of immunoglobulin heavy-chain variable region genes in B-CLL. Whether Zap-70 is a functional target for treatment of the more aggressive CLL cells that express unmutated IgVH is still under investigation.

The use of methylprednisolone (Mp) is widespread in the treatment of B-CLL related concomitant auto-immune disorders. We have recently demonstrated that Zap-70+ B-CLL cells are more resistant to chemotherapy but more susceptible to apoptotic cell death by steroids than Zap-70 negative cells in vitro. We investigated the possible influence of Mp on the expression of Zap-70 in B-CLL cells.

Mononuclear cells of 15 patients with B-CLL were isolated by Ficoll centrifugation and incubated with different concentrations of Mp (range 0.1–500 μM). After 24h and 48h, Zap-70 expression, phospho-ZAP-70 (Tyr 319 and Tyr 493) and apoptosis were evaluated by flow cytometry (indirect staining ZAP-70 and phospho-ZAP-70 and Annexin V-FITC/propidium iodide double staining for apoptosis) and Western blotting when appropriate. Student’s t-test was used for statistical analysis. Mp induces time and dose dependent apoptosis in B-CLL cells (LD50 48h=16.8 μM). After 24h and 48h of incubation at doses of 50 μM, Zap-70 expression decreased significantly (50 μM: p<0.001) and more prominently in B-CLL than in T cells. We also observed a time but not concentration dependent decrease of phospho-ZAP-70 expression, on flow cytometry and western blotting. The observed down-regulation of phospho-ZAP-70 after Mp treatment is probably more important than the down-regulation of ZAP-70, since the phosphorylated form of ZAP-70 is responsible for signal transduction. The effect on P-ZAP-70 can theoretically be explained by an increase in phosphatase activity, a reduction in kinases or an increased susceptibility of the unphosphorylated form to proteasomal degradation. The effect of Mp on P-Zap-70 was inhibitable with the pan-tyrosine-phosphatase inhibitor sodium orthovanadate, but not with the proteasome inhibitor MG0132. Inhibition of the glucocorticoid receptor with mifepristone inhibited induction of apoptosis but not ZAP-70-decrease. Fludarabine treatment of the cells caused comparable levels of apoptosis but negligible decrease of ZAP-70 expression suggesting the decrease in Zap-70 phosphorylation is not due to the apoptosis by itself. On the other hand since mifepristone can inhibit the apoptotic effect of Mp but not the decrease of Zap-70 expression, the potential inhibiting Zap-70 phosphorylation as a therapeutic target should be questioned, and apoptosis induced by methylprednisolone may not depend on it.
Chromosomal Abnormalities in Philadelphia Chromosome (Ph)-Negative Metaphases Appearing during Novel Tyrosine Kinase Inhibitors (NTKI) Therapy in Patients (pts) with Chronic Myeloid Leukemia (CML) after Imatinib-Failure.
Authors: Jabbour Elias , Kantarjian Hagop , Abruzzo Lynne , Giles Francis , Rios Mary Beth , Cortes Jorge
Published: October 2019

The development of chromosomal abnormalities in the Ph-negative metaphases during IM therapy of CML has been recognized in pts with CML. This is different from clonal evolution where the abnormalities are observed in the Ph-positive metaphases. This phenomenon has not been yet systematically assessed to date in patients treated with NTKIs such as dasatinib and nilotinib. By definition, this phenomenon is evaluable only among pts who achieve at least a minor cytogenetic response. We assessed the frequency and the significance of this event among 107 pts with CML after imatinib failure receiving dasatinib (=70) or nilotinib (n=37) therapy between June 2003 and March 2006. After a median follow-up of 13 months (range, 3–32 months), 12 pts (11%) (chronic phase n=6, accelerated phase n=6) receiving dasatinib (n=6) or nilotinib (n=6), developed 21 chromosomal abnormalities in Ph-negative metaphases. Ten (48%) of these abnormalities have been seen in 2 or more metaphases. The median time from the start of the NTKI to appearance of abnormalities was 7 months (range, 0–15 months). The most common cytogenetic abnormalities were: trisomy 8 (n=3, 14%) and del 20q (n=3, 14%). Excluding loss of chromosome Y abnormalities and abnormalities observed in only one metaphase, the incidence was 5%. At the time, abnormalities were detected, 8 pts were in major cytogenetic response (complete in 6) and 4 in minor cytogenetic response. In all but 4 pts these events have been transient and disappeared after a median of 4 months (range, 3–9 months). In 4 pts (+8 n=2, −Y n=1, and add1q, del10q, and del20q n=1), they persisted for a median of 5+ months (range, 3+ – 9+ months). One pt on dastinib developed refractory anemia with ringed sideroblasts (associated with trisomy 8); he had CML for 16 years, failed prior therapy with interferon, imatinib, and nilotinib, and achieved a complete cytogenetic response on dasatinib; none of the other pts has any feature of myelodysplasia. At the last follow-up, all pts are alive: 2 pts lost their response; one of them underwent allogeneic stem cell transplantation and achieved a major molecular remission. The other 10 pts remained in major cytogenetic response (complete in 8). We conclude that cytogenetic abnormalities occur in Ph-negative cells in a small fraction of patients (11%; 5% if loss of Y and abnormalities in one metaphase excluded) treated with the NTKIs. These are frequently transient and usually have no clinical significance, but in rare instances they could signal the emergence of a new malignant clone.
BCR/ABL Promotes Neutrophil Differentiation through Downregulation of c-jun Expression.
Authors: Kobayashi Shinichi , Kimura Fumihiko , Ikeda Takashi , Osawa Yukiko , Torikai Hiroki , Sato Ken , Motoyoshi Kazuo
Published: October 2019

The BCR/ABL fusion protein transforms hematopoietic stem cells and causes chronic myeloid leukemia (CML). An increasing number of mature neutrophils is a characteristic feature in the chronic phase of CML. However, the mechanism by which stem cells transformed by Bcr/Abl differentiate mainly to mature neutrophils remains obscure. To investigate this mechanism, we compared the gene expression profile of CML neutrophils with that of normal neutrophils by microarray analysis. The genes encoding neutrophil granule proteins were upregulated in CML neutrophils, and C/EBPα and C/EBPε, critical transcription factors that regulate granulocytic differentiation, were also upregulated in these neutrophils. On the contrary, the expression of c-jun, a transcriptional factor that contributes to monopoiesis, was downregulated in CML neutrophils. Differences in the expressions of these genes were confirmed by quantitative RT-PCR. A BCR/ABL tyrosine kinase inhibitor, imatinib, released the downregulation of c-jun expression in primary CML neutrophils, showing that Bcr/Abl inhibited the expression of c-jun.

Next, to explore the roles of these transcriptional factors in the chronic phase of CML, we established sublines of KCL22, a cell line derived from CML blastic crisis, in which C/EBPα or C/EBPε expression was inducible (KCL22/α or KCL22/ε respectively). Overexpression of either C/EBP protein resulted in morphological changes, such as a reduction of the nuclear to cytoplasmic ratio, more condensed nuclear chromatin, and segmented nuclei, as well as the expression of differentiation specific markers including G-CSF receptor. These data indicate that C/EBPα/ε expression is sufficient to induce myeloid differentiation in BCR/ABL-positive CML cells. Imatinib treatment released the down regulation of c-jun in KCL22, KCL22/α, and KCL22/ε cells in a manner similar to that in primary cells. Interestingly, imatinib induces monocytic differentiation of KCL22/α cells instead of granulocytic differentiation. This effect of imatinib is independent from C/EBPα induction in KCL22/α cells. The monocytic differentiation and the inhibition of granulocytic differentiation in KCL22/α cells were accompanied by c-jun upregulation. To investigate whether these effects on differentiation of KCL22/α cells depend on releasing the downregulation of c-jun expression with imatinib, we knocked down c-jun expression with siRNA in KCL22/α cells after induction of C/EBPα protein and with imatinib treatment. In the fraction of KCL22/α cells with segmented nuclei, the G-CSF receptor increased when c-jun expression was inhibited with siRNA, indicating that the level of c-jun expression controlled the differentiation fate of CML cells. These findings suggest that Bcr/Abl promotes neutrophil differentiation through downregulation of c-jun accompanied by elevated expressions of C/EBPα/ε.
Imatinib Treatment of Philadelphia Positive (Ph+) Chronic Myeloid Leukemia (CML). For Late Chronic Phase Patients Who Achieve a Complete Cytogenetic Response 5-Year Survival Is as High as for Early Chronic Phase Patients.
Authors: Iacobucci Ilaria , Martinelli Giovanni , Amabile M. , Poerio A. , Soverini S. , Castagnetti F. , Colarossi S. , Gnani A. , Testoni N. , Breccia M. , Abruzzese E. , Martino B. , Cilloni D. , Saglio G. , Pane F. , Rosti G. , Baccarani M.
Published: October 2019

Background: Imatinib (IM) is the drug of choice for the treatment of Ph+ CML, where a complete cytogenetic response (CCgR) is achieved in more than 80% of early chronic phase (ECP) patients, with a 5-year survival of 90% (Simonsson B, Blood 2005). In the patients who initiated the treatment in late chronic phase (LCP) the response rate is lower and the long-term effect on survival is not yet determined.

Aim: We monitored the hematologic, cytogenetic and molecular response to IM in a cohort of 291 patients who were treated with IM in LCP, with focus on progression-free survival and overall survival of complete cytogenetic responders.

Methods: Two hundred and ninety-one patients with Ph+ CML in LCP were enrolled in a national prospective study of the GIMEMA CML Working Party in 2001, and were monitored for cytogenetic response and for molecular response every 6 months. Cytogenetics was performed on bone marrow cells with conventional methods. Molecular response was assessed on bone marrow or peripheral blood samples by quantitative PCR (RQ-PCR) using TaqMan methodology and expressing the results as a ratio of BCR-ABL to ABL x100. Patient age ranged from 19 to 82 years (median 52 years). The duration of chronic phase prior to IM treatment ranged from 1 to 202 months (median 38 months). Treatment was IM 400 mg daily through all the study period, with a few exceptions of dose increase to 600 or 800 mg. The median follow-up time after the first IM dose is 62 months.

Results: One hundred and sixty patients (55%) achieved a CCgR in 3 to 62 months (median 6 months) after the first IM dose and 126 of them (79%) are still in continuous CCgR after 5 years. For the 160 patients the 5-year survival free from progression to accelerated or blastic phase is 95% and overall survival is 91%. These data are very similar to those reported for the ECP patients in the IRIS study (Simonsson B, Blood 2005). At 5 years, 107 patients were evaluated for molecular response; 62% of them were in major molecular response with a BCR-ABL/ABL ratio lower than 0.10. The BCR-ABL transcript was undetectable in 7 cases by RQ-PCR and in 2 cases by nested PCR. No patient developed heart failure.

Conclusions: We confirm that in LCP the CCgR rate to IM is lower than in ECP, but we show that for the complete cytogenetic responders progression-free and overall survival are likely to be as good as for ECP patients, suggesting that the quality of the CCgR is prognostically more important than the duration of leukemia prior to IM treatment.

Figure Figure
Comparison of Combination Therapy of Imatinib with Trisenox Versus Imatinib Monotherapy for Chronic Myeloid Leukemia Patients in Chronic Phase.
Authors: Yang Dongguang , Zhang Ri , Shen Yunfeng , Zhang Xuhui , Wu De Pei
Published: October 2019

Targeted therapy with the Bcr-Abl tyrosine kinase inhibitor Imatinib Mesylate can induce high response rates in chronic myelogenous leukemia (CML) patients. However, evidences that discontinuation of imatinib mesylate inevitably exerts rapid loss of response and some patients with imatinib mesylate monotherapy virtually occur potential relapse suggest that the modification of treatment strategy is critical. We have previously demonstrated that the combination of trisenox (arsenic trioxide) with the tyrosine kinase inhibitor imatinib mesylate or genistein appears to induce markedly more cell apoptosis than imatinib mesylate alone through downregulating Bcl-XL and Bcr-Abl in Bcr-Abl gene transfected HL-60 cells. We here report the preliminary results of a pilot study comparing imatinib mesylate plus trisenox with imatinib mesylate alone for frontline treatment of CML patients.

Up to date 56 patients were enrolled in this clinical trail. All patients (required to be 18 years or older with Bcr-Abl positive CML in chronic phase within three month of diagnosis) were divided into two groups, i.e., monotherapy group and combined therapy group. 42 patients entering monotherapy received imatinib mesylate 400mg daily and 14 patients entering combined therapy imatinib mesylate 400mg daily plus trisenox 10mg daily for one week and then twice a week. We compared treatment results of both groups including complete hematologic response(CHR), major/complete cytogenetic response(MCR/CCR),--defined as 1–35% Ph+ and 0% Ph+ metaphases respectively and major/complete molecular response(MMR/CMR),--defined as ≥ 3 log reduction and negative expression in Bcr-Abl transcript numbers assayed by RQ-PCR respectively. The median follow-up for patients in both groups lasted 36 months. In the combined therapy group, the median age of patients is 42 years old (range, 22–61), the CHR, MCR, CCR, MMR and CMR is 92.8%, 64.3%, 42.9%, 35.7% and 21.4%, respectively. While in the imatinib mesylate monotherapy group, the median age of patients is 46 years old (range, 23–65), the CHR, MCR, CCR, MMR and CMR is 85.7%, 59.5%, 40.5%, 33.3% and 19%, respectively.

Although combination therapy of imatinib with trisenox is not significantly superior to imatinib mesylate monotherapy in efficacy, no resistance case happens in the combination therapy group, in the imatinib mesylate monotherapy group, imatinib resistance occurs in 4 patients. In addition, the safety and tolerability of a combination of imatinib mesylate and trisenox is good. This result indicates that the combination of imatinib and trisenox to treat chronic myeloid leukemia may be promising in avoiding the occur of imatinib resistance.
Dasatinib (SPRYCEL®) in Patients (pts) with Previously Untreated Chronic Myelogenous Leukemia (CML) in Chronic Phase (CML-CP).
Authors: Cortes Jorge , O’Brien S. , Jones D. , Borthakur G. , Giles F. , Nicaise C. , Kantarjian H.M.
Published: October 2019

Dasatinib (SPRYCEL®, formerly BMS-354825) is a multi-targeted kinase inhibitor of BCR-ABL and SRC. Based on its high level of activity, leading to FDA approval in pts with CML-CP who are resistant to or intolerant of imatinib (im), the present phase II trial was designed to study previously untreated CML-CP pts treated with dasatinib. The primary objective was to estimate the proportion of pts attaining major molecular response (BCR-ABL/ABL ratio ≤0.05% by qPCR) at 12 months (mo). All pts received dasatinib orally 100 mg/day, and were randomized to either a 50 mg-twice-daily or a 100 mg-once-daily schedule. Dose escalation to 140 mg/day and 180 mg/day for poor response or dose reduction to 80 mg/day and 40 mg/day for toxicity, maintaining the same schedule, was allowed. Blood counts were done weekly for the first 4 weeks (wks), every 4–6 wks for the first year, and then every 3–4 mo; bone marrow aspirates with cytogenetics were obtained at baseline and every 3–4 mo during the first year, and then every 6–12 mo; molecular monitoring of BCR-ABL transcript levels by qPCR was done at baseline, every 3-4 mo for the first year, and then every 6–12 mo. Of the first 24 pts enrolled between November 2005 and June 2006, pooled across dosing schedule, 54% were female; median age was 44 years (range 18–76). Nine (38%) of the pts were Sokal intermediate-risk and 1 (4%) was high-risk. Median baseline WBC count was 17.4 x109/L (range 3.7–300.0). At 3 mo, complete hematologic response (CHR) and major cytogenetic response (MCyR) was seen in 17 (89%) of 19 pts who had received at least 3 mo of therapy, and complete cytogenetic response (CCyR) was seen in 15 pts (79%). This compares favorably with a CCyR at 3 mo of 37% with im 400 mg/day and 61% with im 800 mg/day, in historical data of similar patients treated in studies at MD Anderson. qPCR at 3 months was <1% (ie, approximately 2-log reduction) in 5 (26%), and was <10% in 9 (47%) of these 19 pts. The most common non-hematologic adverse events (AE) included dyspnea (8 pts), fatigue (7 pts), muscle pain (6 pts), and headache (5 pts) and were predominantly grade (gr) 1–2. Pleural effusion occurred in 3 pts and was gr 1–2 in all. Hematologic toxicity included anemia in 8 pts (4 gr 3), pancytopenia in 4 pts (3 gr 3, 1 gr 4), and thrombocytopenia in 4 pts (2 gr 3, 2 gr 4). With a median duration of therapy of 5 mo, there were 10 pts who required interruption of treatment, 6 due to non-hematologic toxicities, 2 due to hematologic toxicities, and 2 due to both. Dose reductions occurred in 6 pts, 3 due to non-hematologic toxicity, 1 due to hematologic toxicity, and 2 due to both. Rapid, complete cytogenetic responses to dasatinib 100 mg/day have been observed in a high percentage of patients with previously untreated CML-CP. Accrual to this trial continues, and updated efficacy and safety data will be presented at the meeting.
Combination of the Histone Deacetylase Inhibitor Vorinostat and Dasatinib Increases Apoptosis in Bcr-abl+ Cells and Reverses Changes Associated with CML Progression.
Authors: Kretzner Leo , Scuto Anna , Juhasz Agnes , Jove Richard , Yen Yun , Forman Stephen J. , Kirschbaum Mark
Published: October 2019

Chronic myelogenous leukemia (CML) is associated with the bcr-abl fusion gene product, a constitutively active non-receptor tyrosine kinase driving cell division. The potent bcr-abl kinase inhibitor, dasatinib, is now FDA approved for imatinib-refractory patients, being active in most cases other than those with the T315I mutation. However, accelerated phase CML is typified by further genetic changes, including the suppression of various DNA damage response and apoptosis related proteins (Radich, et al PNAS 2006). Thus a novel approach which may reverse these changes, might provide better treatment of accelerated phase CML.

A new class of agents targeting epigenetic processes, the histone deacetylase inhibitors (HDACi), is believed to act upon chromatin allowing re-expression of tumor suppressor genes believed to be closed off to transcription by the tumor in its effort to grow in an uncontrolled manner. We reasoned that adding an HDACi to dasatinib treatment of CML cells could promote re-expression of genes responding to genetic instability in these cells. To test this hypothesis we tested dasatinib (D) and the HDACi Vorinistat (V, suberoylanilide hydroxamic acid, SAHA) alone and in combination, using K562 cells. Apoptosis was measured by Annexin V staining after 48 hours.

Apoptosis results: 1uM Vorinostat: 10%; 2 uM V: 16%. Dasatinib 0.5 nM: 28%, 1 nM D: 44% In combination, 1uM V + 0.5 nM D: 41%, 1 uM V + 1nM D: 64%, 2 uM V + 0.5 nM D: 65%, 2 uM V + 1 nM D: 67%, suggesting significant increase in apoptosis for the combination over either single agent treatment alone.

We therefore began surveying panels of DNA damage- and apoptosis-related genes by means of RT-PCR. Studying the combination of 2 uM V + 1 nM D, we found increased expression of several proteins, including GADD45G and FANCG, both DNA damage response proteins suppressed in the progression to accelerated phase CML. Other proapoptotic proteins were increased such as MAP2K6, SEMA4A, BIK, and TNF superfamily members 7 and 25. This data suggests that the changes associated with progression from chronic phase to accelerated and blast crisis CML may be epigenetic in nature, and that these changes may be reversed by the combination of vorinostat and dasatinib. A clinical trial of dasatanib and vorinostat in advanced phase CML would be of value.
A Phase II Study of Nilotinib A Novel Tyrosine Kinase Inhibitor Administered to Imatinib-Resistant or Intolerant Patients with Chronic Myelogenous Leukemia (CML) in Accelerated Phase (AP).
Authors: Kantarjian Hagop M. , Gattermann Norbert , Hochhaus Andreas , Larson Richard , Rafferty Teresa , Weitzman Aaron , Haque Ariful , Giles Francis , O’Brien S.G. , le Coutre Philipp
Published: October 2019

Nilotinib is a potent, highly selective, aminopyrimidine inhibitor of Bcr-Abl which in vitro is 30-fold more potent than imatinib. It is active against 32/33 imatinib resistant Bcr-Abl mutations. This open-label study was designed to evaluate the safety and efficacy as defined by hematologic/cytogenetic response (HR/CyR) rates of nilotinib at a dose of 400 mg bid in imatinib resistant or intolerant AP patients. Daily doses of nilotinib could be escalated to 600 mg BID for patients who did not adequately respond to treatment, and in the absence of safety concerns. Safety and efficacy data are reported for 64 patients of which 52 (81%) are resistant and 12 (19%) are intolerant to imatinib. More than half (63%) of the patients had CML for ≥ 5 years. The median age was 61 (range 24-79) years and the median time from CML diagnosis and AP diagnosis were 74 (range 2 to 298), and 2 (range 0-106) months, respectively. Of the 64 patients, 17 (27%) had extramedullary disease at baseline. The median duration of nilotinib exposure was 141 (range 2–380) days and the median average dose intensity (mg/days) for all patients, with and without dose escalations, was 797 (range 157 to 1136). Treatment is ongoing for 33 (52%) patients, and 31 (48%) have discontinued (14 for disease progression, 8 for adverse events, 1 each for an abnormal laboratory value, administrative problems, lost to follow up, 4 patients withdrew consent and there were 2 deaths listed as the primary reason for discontinuation). Overall, there were 7 deaths including 4 for disease progression, one related to progressive disease complicated by a cerebral hemorrhage, one cardiac failure and one due to sepsis. Confirmed HR occurred in 28 (44%) patients, of which 11 (17%) were complete, 5 (8%) were marrow responses/no evidence of leukemia, 12 (19%) were return to chronic phase. There were 7 (11%) patients with stable disease/no response, 6 (9%) with disease progression and 21 (33%) patients were not evaluable. Major CyR occurred in 20 (31%) patients, of which 11 (17%) were complete, 9 (14%) were partial, 11 (17%) were minor, and 15 (23%) were minimal.

There were 6 patients (9%) that did not respond. The rate of major CyR for the resistant and intolerant patients was 16 (31%) and 3 (25%), respectively. The majority of Grade 3 or 4 adverse events included thrombocytopenia in 21 (33%), neutropenia in 17 (27%), anemia in 10 (16%) patients, decreased hemoglobin in 4 (6%) patients, and increased lipase in 5 (8%). In summary, these data suggest nilotinib is clinically active and has an acceptable safety and tolerability profile when administered to patients with CML-AP. Updated information will be presented at the meeting.
BH3 Mimetic Augments the Induction of Apoptosis by INNO-406, a Novel Bcr-Abl/Lyn Inhibitor, in Bcr-Abl+ Leukemic Cells.
Authors: Kuroda Junya , Kimura Shinya , Strasser Andreas , Andreeff Michael , Ashihara Eishi , Tabe Yoko , Taniwaki Masafumi , Maekawa Taira
Published: October 2019

Aberrant tyrosine kinase activity of Bcr-Abl fusion oncoprotein by Philadelphia chromosome translocation is the causative as well as the primary molecular target in most of CML and approximately 20% of adult ALL. Though imatinib mesylate has improved the treatment of Bcr-Abl+ leukemia, resistance is often reported in patients with advanced-stage disease. To override imatinib-resistance mechanisms, we have developed INNO-406 (formerly NS-187; Kimura et al, Blood 2005), a novel Bcr-Abl/Lyn inhibitor that is 25~55-folds more potent than imatinib in suppressing Bcr-Abl+ leukemias, and is active against most of imatinib-resistant mutated Bcr-Abl except T315I. Phase I study on INNO-406 started in July 2006. To date, there is no data on the clinical-resistance profile of INNO-406. However, some patients may become resistance after the prolonged administration of INNO-406, as with other Abl kinase inhibitors such as dasatinib and nilotinib. A combination of targeted drugs with different specificities might overcome the problem of resistance (Komarova & Wodarz, PNAS 2005). Thus, we tried to find the best partner for INNO-406 based on the apoptosis-induction mechanisms of each agent. We found that apoptosis induced by INNO-406 was mediated by a Bcl-2 family-regulated intrinsic apoptosis pathway and required the activation of several BH3-only proteins such as Bim, Bad, Bmf or Bik. We investigated the combined effects of 17-AAG or bortezomib, both agents those are known to increase Bim and/or other BH3-only proteins. Neither 17-AAG nor bortezomib showed any additive effects on the accumulation of BH3-only proteins compared with INNO-406 alone. This finding suggested that INNO-406 alone activated BH3-only proteins maximally and combined effects for induction of apoptosis via the activation of BH3-only proteins could not be expected. Thus, we next examined the effects of ABT-737 (produced by Michael Andreeff), which was not an inducer of BH3-only proteins but an inhibitor for anti-apoptotic Bcl-2/Bcl-XL proteins. Intriguingly, ABT-737 augmented significantly the induction of apoptosis by INNO-406 not only in wild-type but also mutated Bcr-Abl+ leukemic cells (Figure). In conclusion, the combined therapy with a Bcr-Abl inhibitor, such as INNO-406, which induces BH3-only proteins strongly and an inhibitor for anti-apoptotic Bcl-2/Bcl-XL proteins such as ABT-737 might be the most rationale apoptosis-based molecular targeting approach against Bcr-Abl+ leukemias.

Figure Figure
Imatinib-Resistant BCR-ABL Mutant Cellular Screening Paradigm To Characterize Promising Small-Molecule Inhibitors: T315I-BCR-ABL Targeted Drug Discovery.
Authors: Azam Mohammad , Shakespeare William C. , Metcalf Chester , Wang Yihan , Sunderamoorthi Raji , Zhu Xiaotan , Huang Wei-Sheng , Zou Dong , Thomas Mathew , Romero Jan , Keats Jeff , Xu Qihang , Dalgarno David , Clackson Tim , Sawyer Tomi K. , Daley George Q.
Published: October 2019

In patients with chronic myeloid leukemia (CML), kinase domain mutations account for imatinib resistance in the majority of cases. Mutations cause either a direct steric hindrance to drug binding or a conformational change that favors kinase activation, which therefore precludes imatinib binding. We have previously characterized the dual Src-Abl kinase inhibitor AP23464 and found it to effectively suppress the growth of cells expressing native and essentially all imatinib-resistant variants of BCR-ABL, with the notable exception of the gatekeeper T315I mutant (Azam et al., Proc. Natl. Acad. Sci. USA, 103: 9244, 2006). Following this work, we have used mutant panel screening and integrated structural analysis to further characterize key analogs designed to overcome T315I resistance, as exemplified by AP23846 and AP24163. Both molecules effectively inhibit the tyrosine kinase activity of wild type (WT) and T315I variants of BCR-ABL, and inhibit the proliferation of BaF3-derived cell lines expressing these enzymes (see Table below). AP24163 was further characterized against a broader panel of imatinib-resistant BCR-ABL-expressing cell lines and showed a promising profile of proliferation inhibition. Comparison of these data with structural models of the mutants provides insights into the basis for the ability of AP24163 to overcome imatinib resistance.

Refinement of small-molecule kinase inhibitors by the integration of sequential screening of panels of mutants coupled with structural analysis is a powerful drug discovery paradigm that is applicable to an increasing number of targeted therapeutic agents.

INHIBITION OF PROLIFERATION OF BAF3 CELLS EXPRESSING BCR-ABL AND ITS VARIANTS (IC50 in nM) IMATINIB AP23464 AP23846 AP24163 WT 600 14 500 7 T315I >20000 >1000 500 480 L248R >20000 92 ND 64 G250E 5000 25 ND 63 Q252H 3000 40 ND 42 Y253H 18000 32 ND 44 E255K 12000 74 ND 24 BAF3+IL3 >20000 >1000 500 >10000 Figure Figure
In Vitro and In Vivo Inhibition of the T315I Mutant BCR/ABL Kinase.
Authors: Zhu Hong , Hanna Ehab , Lohse Dan , Stoughton Silva , Tam Betty , Barrett Kathy , Noronha Glenn , Dneprovskaia Elena , Cao Jon , Chow Chun , Hwang Linda , Mak Chiching , McPherson Andy , Palanki Moorthy , Pathak Ved P. , Renick Joe , Soll Richard , Zeng Binqi , Hood John
Published: October 2019

The T315I point mutation situated at the ATP-binding site of BCR/ABL tyrosine kinase remains to be the major unmet medical need in treating CML patients. None of the marketed CML drugs, including Imatinib or the recently approved Dasatinib, are able to inhibit this clinically most prevalent mutant of BCR/ABL kinase. To overcome the T315I mutation-presented hurdle, a novel small molecule BCR/ABL inhibitor, TG101223, has been synthesized and characterized in vitro and in vivo. Enzymatic activity of T315I mutant kinase and proliferation of BaF3:BCR/ABLT315I cells were inhibited with an IC50 of 50 nM and EC50 of 500 nM respectively. However, little effect of TG101223 was observed on the proliferation of the control BaF3 cells (IC50≥5,000 nM). Consistent with these observations, 24 hour treatment with 2,000 nM TG101223 induced DNA fragmentation in BCR/ABLT315I cells, but not in the control BaF3 cells. Furthermore, caspase-3 activation was detected in BCR/ABLT315I cells by an in-cell caspase assay following 4 h treatment with 2,000 nM TG101223. The above results strongly suggest that TG101223 inhibits BCR/ABLT315I-mediated cell proliferation and survival. In contrast, Imatinib did not inhibit AblT315I enzyme nor BCR/ABLT315I cell proliferation. The kinase selectivity of TG101223 was determined using a commercial enzyme assay with a panel of 49 phylogentically diverse protein kinases. Only Abl, Flt3 and the mutants of those two kinases were inhibited by more than 80% with 500 nM TG101223. Importantly, TG101223 demonstrated a desirable pharmacokinetic profile in mouse (oral bioavailability: 40%, clearance: 23 mL/min/kg and T½: 5 h). The tumorogenicity of BCR/AblT315I cells were confirmed by the observation that tail vein infusion with BCR/ABLT315I cells induced splenomegaly in SCID mice and death by day 14. To assess the in vivo activities of TG101223, mice were dosed orally once with TG101223 on day 13, followed by harvesting of their spleens 7 hours after dosing for Western blot-based protein phosphorylation analysis. A significant induction in phosphorylation of both CrkL and BCR/ABL was observed in the spleens of T315I cell recipient mice, supporting the notion that splenomegaly is a result of the infiltration of proliferating BCR/ABLT315I cells. The above induction in spleen CrkL and BCR/ABL phosphorylation was inhibited by TG101223. These data suggest that TG101223 is a promising candidate for treating the drug resistant CML patients.
Prognostic Significance of Minimal Residual Disease (MRD) in Childhood B-Precursor ALL and Its Relation to Other Risk Factors. A Children’s Oncology Group (COG) Study.
Authors: Borowitz Michael J. , Devidas Meenakshi , Bowman W. Paul , Carroll William L. , Chen I.-Ming , Harvey Richard , Hunger Stephen , Martin Paul L. , Pullen Jeanette , Viswanatha David , Willman Cheryl L. , Winick Naomi , Camitta Bruce
Published: October 2019

Early response to therapy, including assessment of MRD is a powerful prognostic factor in childhood ALL. However, there are many other known prognostic factors, and the precise relationship among these and MRD has been incompletely explored. In COG protocol P9900, patients were assigned to one of 3 risk-directed therapies based on NCI risk group, as well as TEL-AML1 translocations (TEL) and simultaneous trisomies of chromosomes 4 and 10 (DT). Infants and Ph+ patients were excluded. Good risk patients (9904) comprised NCI SR patients with either TEL or DT without CNS disease. Poor risk patients (9906) included a subset of NCI HR patients with particularly unfavorable risk factors based largely on age and white count, while 9905 had both NCI SR and HR patients not eligible for 9904 or 9906, including NCI HR patients with TEL or DT. MRD was satisfactorily measured with at least .01% sensitivity by 4-color flow cytometry in the marrow at end of induction (D29) or in blood at Day 8 (D8). D29 MRD was associated with poor outcome in all studies, and in both HR and SR patients on 9905 as shown:

Study 4 y EFS ± SE (n) p value MRD <.01% MRD >.01% 9904 Overall 94±1%(668) 75±6%(105) <.0001 TEL-AML1 94±2%(367) 68±12%(38) <.0001 DT 95±1%(316) 80±7%(69) <.0001 9905 NCI-Standard Risk 89±2%(462) 63±6%(119) <.0001 9905 NCI-High Risk 83±4%(292) 49±9%(73) <.0001 9906 74±6%(156) 39±8%(84) <.0001

The proportion of D29 MRD positive DT(17.9%) patients was higher than that of TEL patients (9.4%) but MRD was also prognostic when each group was considered separately; similar findings were seen in HR TEL and DT patients. TEL or DT was associated with a significantly better prognosis in both HR and SR patients who were MRD negative, but were not favorable factors in MRD positive patients. D8 blood MRD was also prognostic in all studies, with higher MRD levels generally associated with poorer outcome. Patients who were both D29 and D8 MRD negative had a good outcome overall (93% 4 y EFS); combining all favorable risk factors, including TEL, DT, NCI risk group, D8 and D29 MRD identified a group of patients (about 13% of B-precursor ALL patients) with 98± 1% 4 y EFS. In multivariate analysis, D29 MRD was the most important prognostic factor. NCI Risk group, DT and D8 blood were also significant, but TEL and day 8 morphologic assessment of marrow were not. We conclude that MRD is a major prognostic factor in childhood ALL even accounting for other risk factors. The prognostic significance of TEL and DT depends on MRD status. The combination of good risk clinical and genetic factors, and absence of MRD in both D8 blood and D29 marrow identifies a group of patients with outstanding outcome on limited therapy.
Mutations in the BCR/ABL Kinase Encoding the Resistance to Imatinib Mesylate Do Not Modify the Sensitivity to Genotoxic Treatment.
Authors: Bargaoanu Milene , Skorski Tomasz
Published: October 2019

Imatinib mesylate (IM), a selective inhibitor of ABL kinase activity, revolutionized the treatment of BCR/ABL-positive leukemias. Unfortunately, clinical and experimental observations reveal that resistance to IM is a rising problem, which obscures an otherwise very successful oncogene-targeted therapy. IM resistance can be achieved by point mutations in the kinase domain of BCR/ABL and have been detected in 50–90% of patients with acquired resistance to IM, including ~23% of the IM-naive patients. Strategies to enhance the effect of IM and eventually overcome the resistance are dose escalation, addition of a growth factor, and combinations with novel tyrosine kinase inhibitors like AMN107 and dasatinib, or with inhibitors targeting downstream BCR/ABL effectors, e.g. PI-3k. Unfortunately, resistance to other small molecule inhibitors is likely to appear, as well. Therefore, we tested the sensitivity of leukemia cells expressing IM-resistant BCR/ABL mutants to genotoxic agents. Baf3 cells expressing similar levels of p210BCR/ABL wild-type (WT) and Y253F, Y253H, E255K, E255V, T315I, M351T, and H396P mutants were established. These mutants were selected since they represent several functionally distinct ABL kinase domain regions, including P-loop (Y253F/H and E255K/V), the site of a hydrogen bond with IM (T315I), the activation loop hinge (M351T), and the activation loop (H396P). In addition, they exhibit altered transformation potency, kinase activity, and substrate utilization, irrespective of sensitivity to IM. Western analysis demonstrated similarity, but also differences in the patterns of tyrosine phosphorylated proteins in total cell lysates. As expected, these clones displayed different level of sensitivity to IM: T315I>Y253H>E255V>E255K=Y253F>M251T=H396P>WT. However, leukemia cells expressing the WT and IM-resistant BCR/ABL kinase mutants demonstrated similar sensitivity to cytotoxic drugs such as hydroxyurea (HU), mitomycin C (MMC), and N-Methyl N’-nitro-N-nitrosoguanidine (MNNG). Surprisingly, clones expressing IM-resistant BCR/ABL kinase mutants were more sensitive to γ-irradiation than their WT counterparts. γ-H2AX (histone H2AX phosphorylated on S139) nuclear foci, which serve as marker of DNA double-strand breaks (DSBs), the most lethal DNA lesions, were detected by immunofluorescence. Leukemia cells expressing WT and IM-resistant p210BCR/ABL proteins accumulated similar numbers of DSBs after MMC treatment and γ-irradiation. This observation suggests that leukemia cells expressing IM-resistant p210BCR/ABL mutants may be eliminated by genotoxic treatment, especially γ-irradiation. In addition, IM-resistant BCR/ABL kinase mutants, in contrast to the WT kinase may not be able to modulate the mechanisms protecting from apoptosis induced by γ-irradiation.