bluuebunny/crossref_metadata_2025 huggingface dataset:
Preliminary Activity of Nilotinib (AMN107), a Novel Selective Potent Oral Bcr-Abl Tyrosine Kinase Inhibitor, in Newly Diagnosed Philadelphia Chromosome (Ph)-Positive Chronic Phase Chronic Myelogenous Leukemia (CML-CP).
Authors: Jabbour Elias , Cortes Jorge , Giles Francis , O’Brien Susan , Letvak Laurie , Kantarjian Hagop
Published: October 2019

Nilotinib is a novel, highly selective oral Bcr-Abl inhibitor which is approximately 30-fold more potent than imatinib. High response rates with nilotinib were observed in all CML phases post imatinib failure. We evaluated the efficacy of nilotinib in newly diagnosed Ph-positive CML-CP. Thirteen patients with newly diagnosed Ph-positive CML-CP were treated with nilotinib 400 mg orally twice daily. The median age was 49 years (range, 24–72 years). The Sokal risk at pretreatment was low in 10 patients, intermediate in 2, and high in 1. The median follow-up is 8 months (range, 3–12 months). All patients have reached the 6-month evaluation. The rate of complete cytogenetic response [CGCR] (Ph 0%) at 3 and 6 months was 93% and 100%, respectively. This is compared with a CGCR at 3 months of 37% and with imatinib 400 mg/d and 61% with imatinib 800 mg/d (p=0.0002) and 54% and 85% at 6 months, respectively (p<0.0001), in historical data of newly diagnosed patients treated in studies at M. D. Anderson. Six patients were evaluable at 9 months and all were in CGCR. The median QPCR with nilotinib at 3, 6, and 9 months were, respectively, 3.4% (range, 0.02–29.5%), 1.8% (range, 0.004–9.13%), and 0.54% (range, 0.04–1.28%). At 3-month follow-up, major molecular response (BCR-ABL/ABL ratio<0.05%) was observed in 1/13 patients (8%) and in 6/11 (55%) at 6-month. Grade 3–4 myelosuppression was observed in 3 of the 13 patients and other grade 3–4 side effects in 3 patients (increased lipase in 2 and musculo-skeletal pain in 1). Four patients had their dose reduced to 400 mg daily due to extramedullary toxicity. Two patients were taken off after 6 and 8 months (patient choice) and switched to imatinib. In conclusion, early results with nilotinib 400 mg orally twice daily suggest significant efficacy manifested by complete cytogenetic responses in nearly all patients as early as 3 months after the start of therapy with a favorable toxicity profile.
Gene Expression Profiling of Paired Pre- and Post-Prednisolone (PRED) BM Samples from Childhood ALL Identifies Robust Signatures for PRED Response and Eventual Outcome.
Authors: Lu Yi , Liu Huiqing , Xu Ying , Lin Koh Pei , Hany Ariffin , Wong Limsoon , Yeoh Allen Eng Juh
Published: October 2019

Early response to therapy is the most important prognostic factor for childhood ALL. CCG investigators have shown that Day-7 and Day-14 BM blast counts were prognostically important although there is great inter-observer variability. BFM group have shown that day 8 prednisolone (PRED) response is highly predictive of the treatment outcome. While gene expression profiling (GEP) of diagnostic marrow can discern a pattern of PRED sensitivity as determined by in vitro MTT assay, the accuracy was low at only 70%. We hypothesized that changes in global GEP after therapy have a higher likelihood to predict response as the signatures of sensitivity and resistance may be unmasked during the therapy. We prospectively studied the changes in GEP using Affymetrix HG-U133A or Plus 2 chips on paired BM samples before and after 7-day course of PRED and one dose IT MTX in 58 patients with newly diagnosed or relapsed ALL. Unsupervised hierarchical clustering revealed that pre- and post- PRED samples in the patients still tended to cluster together, indicating that expression profiles of molecular subgroups were still most important. To remove intrinsic influence of molecular subtypes and identify potential signatures independent of genetic abnormalities, we subtracted Day-0 GEP from its paired Day-8 profile and retained probe sets with significant changes (≥ 10-fold). To avoid the ambiguity of variation in BM blast counting at Day-8, we divided the samples into a stringently reproducible group where “Good” PRED response was defined as that Day-8 blast count in PBL < 109/L and BM lymphoblasts ≤ 30% (n=16). “Poor” response was when Day 8 PBL ≥ 109/L (n=11). This stringently reproducible group (n=27) formed the training group to help define a distinct signature while the rest (n=31 pairs) were used as a blinded test set. 54 and 19 discriminating genes were identified by 2 independent statistical methods respectively, and an integrated predictor model was constructed based on shortlisted entries. This model predicted the PRED response with 100% accuracy for the training set using the leave-one-out cross validation but was less accurate in predicting the BM blast count in blinded test set. But intriguingly, in the blinded test set, this model predicted correctly 19 out of 21 reliable “Good” PRED responses are in CCR (91%), while among 8 predicted as “Poor” responses, only 2 are in CCR (25%). This suggests that as gene expression profiling as early as day 8 of PRED could discern the beginning of leukaemia cell death even before morphological changes are discernable and is highly correlated to eventual outcome. In conclusion, we have shown that analyses on the relative changes of gene expression profile can identify real genetic signatures indicating the sensitivity to PRED administration which is highly correlated with outcome.
Mantle Cell Lymphoma: An Epigenetic Disease of Cyclin D1 Deregulation Involving Nucleophosmin, CTCF and Small RNA’s.
Authors: Epner Elliot , Jing Huang , Jin Wang , Fleming W.H.
Published: October 2019

Translocations involving the immunoglobulin heavy chain gene locus (IgH) are common in B cell malignancies. The molecular mechanisms of long distance gene activation by IgH regulatory elements in B cell malignancies are unknown. One common target gene is cyclin D1, which is deregulated in most patients with mantle cell lymphoma (MCL) and 20–30% of patients with multiple myeloma (MM). Cyclin D1 is known not to be expressed in normal lymphocytes, yet is deregulated by IgH translocations and insertions. Chromatin immunoprecipitation (ChIP) assays demonstrated the presence of the CCCTC-binding factor (CTCF) at both the cyclin D1 promoter and 3′ IgH regulatory regions only in cyclin D1 expressing malignant B cell lines containing IgH translocations or insertions. The nucleolar protein nucleophosmin (NPM), a newly identified CTCF interaction partner, exhibited a similar distribution in ChIP assays. Combined FISH/immunofluorescence assays in MCL cell lines and patient samples have demonstrated the presence of the cyclin D1/IgH translocated locus at the nucleolus specifically in cells with the t(11;14). Short hairpin (Sh) RNA mediated NPM knockdown results in a specific growth arrest only in cells with translocated chromosomes. Cell growth in variant MCL cells and B lymphocytes is not altered. The presence of small RNA’s and binding of the protein Dicer at the translocation breakpoint has been demonstrated. ShRNA knockdown of dicer results in significant reduction of cyclin D1 protein in MCL cells. Tethering of the cyclin D1 promoter to the 3′ IgH regulatory regions at the nucleolar membrane by CTCF/nucleophosmin provides a mechanism for spatial juxtaposition of the cyclin D1 promoter and 3′ IgH regulatory elements that facilitates deregulated expression of the cyclin D1 gene. These results also demonstrate the participation of small RNA’s and the RNA interference pathway in the deregulated expression of cyclin D1 in MCL.
Fyn Upregulation Is a Novel ROS-Dependent Mechanism Controlling CML Growth, Progression and Imatinib Resistance.
Authors: Chandra Joya , Amin Hesham M. , Howard Adrienne , Miller Claudia P. , Lin Quan , Ban Kechen
Published: October 2019

The BCR/ABL kinase alters the oxidative environment in chronic myelogenous leukemia (CML) cells, but the consequences of the increased reactive oxygen species (ROS) levels on signaling pathways remain unknown. Increased intracellular peroxides in BCR/ABL expressing cells have been linked to DNA damage, which may promote blast crisis in CML. We report that Fyn is a BCR/ABL target that is upregulated in an oxidant- sensitive manner. Cells overexpressing BCR/ABL display a four-fold upregulation of Fyn protein, which is blocked by chemical antioxidants. This increase in Fyn directs proliferative and survival signals since knockdown of Fyn using shRNA slows leukemia cell growth by 50% both in vitro and in vivo, inhibits clonogenic growth by 45% and leads to increased sensitivity to imatinib. Jak2 inhibition prevents Fyn protein upregulation, suggesting that Jak2 is upstream of Fyn, and we indeed find that Jak2 levels are increased in BCR/ABL expressing cells. In a cohort of CML patients Fyn expression was significantly increased in blastic phase CML samples as compared to chronic phase, confirming the clinical relevance of Fyn upregulation. Collectively, these results demonstrate that oxidant-dependent, Jak2-dependent upregulation of Fyn is a novel alteration in CML that is critical for cell growth and imatinib resistance.
Human T-Cell Leukemia Virus Type I Tax Activates CD69 Gene Expression.
Authors: Ishikawa Chie , Okudaira Taeko , Nakazato Tetsuro , Tomita Mariko , Mori Naoki
Published: October 2019

The human T-cell leukemia virus type I (HTLV-I) is an oncogenic retrovirus that is etiologically linked to the genesis of adult T-cell leukemia (ATL). Emerging evidence suggests that the pathogenicity of ATL involves suppression of the overall immune response, although the underlying mechanism remains unclear. In this study, we demonstrated that HTLV-I transactivator Tax induces the aberrant expression of CD69, an early leukocyte activation molecule that plays an important role in downregulation of the immune response. In a panel of HTLV-I-infected T-cell lines, CD69 expression was highly elevated compared to HTLV-I-negative T-cell lines at both mRNA and protein levels. Furthermore, CD69 expression correlated with Tax expression. Peripheral blood mononuclear cells from ATL patients also showed an increased expression of CD69 compared with controls. In vitro infection of a T-cell line with HTLV-I was associated with CD69 expression in conjunction with the increasing Tax expression. Expression of CD69 was dependent upon Tax expression in the inducible Tax-expressing cell line JPX-9. Tax transactivated the CD69 gene promoter in a transient transfection assay. Using Tax mutants and dominant negative mutants of IκBs, IKKs, NIK, and CREB, we demonstrated that Tax-induced CD69 expression required the NF-κB and CREB signaling pathways. A series of deletion and mutation analyses of the CD69 gene promoter indicated that two NF-κB, two EGR, and a CRE sequences were critical for Tax transactivation. Electrophoretic mobility shift assay showed the formation of specific protein-DNA complexes in HTLV-I-infected T-cell lines. These results suggest that Tax directly transactivated CD69 gene expression, through multiple cis-acting elements and by the interplay of transcription factors of the NF-κB, EGR, and CREB families. Tax-induced CD69 expression may be involved in immune suppression in ATL.
Clinical Relevance of Complex Chromosomal Aberrations in Bone Marrow Cells of 107 Children with ETV6/RUNX1 Positive Acute Lymphoblastic Leukemia (ALL).
Authors: Zemanova Zuzana , Michalova Kyra , Babicka Libuse , Pavlistova Lenka , Jarosova Marie , Holzerova Milena , Oltova Alexandra , Hruba Martina , Muzikova Katerina , Zuna Jan , Trka Jan , Mihal Vladimir , Sterba Jiri , Formankova Renata , Sedlacek Petr , Vrzalova Alena , Stary Jan
Published: October 2019

Cryptic translocation t(12;21)(p13;q22) which give origin to the ETV6/RUNX1 hybrid gene can be found by I-FISH in approximately 20–25% of children with B precursor ALL as the most frequent specific aberration. This translocation is generally associated with good outcome. Despite of its favorable prognostic value, late relapses may occur within this group of patients. One of the reasons could be the high instability of the genome of leukemic cells, which is manifested at the chromosomal level by additional aberrations and/or complex chromosomal rearrangements. The aim of the study was to evaluate the significance of the additional chromosomal aberrations for prognosis of children with ETV6/RUNX1 positive ALL.

For the assessment of ETV6/RUNX1 fusion gene RT-PCR and/or double target interphase FISH with locus-specific probe (Abbott-Vysis, Des Plaines, Illinois, USA) were used (200 interphase nuclei analyzed, cut-off level 2.5% tested on controls, standard deviation ≤0.5%). Karyotypes were analyzed by conventional and molecular cytogenetic methods. Structural and/or complex chromosomal aberration were verified by FISH with whole chromosome painting probes (Cambio, Cambridge, UK) and/or by mFISH with the "24XCyte" probe kit (MetaSystems GmbH, Altlussheim, Germany).

We performed prospective and retrospective study of 107 children with ALL and ETV6/RUNX1 fusion gene detected by RT-PCR and/or I-FISH. Patients were diagnosed between 1995 and 2006, age ranged between 15 months and 16.9 years (median 4.2 years). Relapse appeared in 19 children (17.8%), four of them died. In 64 children (59.8%) we found besides t(12;21)(p13;q22) additional chromosomal aberrations, the most frequently trisomy or tetrasomy of chromosome 21 (20 cases), deletion of non-translocated ETV6 allele (24 cases), deletion of 6q (7 cases) and/or rearrangements of the long arm of chromosome X (6 cases). Complex karyotypes were identified in 38 children (35.5%). In twelve of them variant translocations of chromosomes 12 and 21 with other partners were observed. Event-free survival (EFS) was significantly shorter in patients with additional structural and/or complex aberrations in ETV6/RUNX1 positive cells (p=0.01).

In our cohort complex karyotypes indicated poor prognosis. Finding of complex chromosomal aberrations in leukemic cells is accompanied by higher risk of relapse even in those cases where the prognostically positive aberration is primarily present.
IgA and IgG Hypogammaglobulinemia Are Associated with Mutations in the APRIL/BLYS Receptor TACI in Waldenstrom’s Macroglobulinemia (WM).
Authors: Hunter Zachary , Leleu Xavier , Hatjiharissi Evdoxia , Santos Daniel , Xu Lian , Ho Alan , Bakthavachalam Vinod , Moreau Anne-Sophie , O’Connor Kelly , Soumerai Jacob , Patterson Christopher , Ghobrial Irene , Hamilton Susanna , Verselis Sigitas , Fox Edward , Treon Steven
Published: October 2019

IgA and IgG hypogammaglobulinemia are a common finding in patients with WM which may contribute to recurrent infections seen in up to half of WM patients. The etiology for this finding remains unclear. As part of these studies, we investigated the prevalence and genetic basis for IgA and IgG hypogammaglobulinemia among 89 previously untreated patients with the clinicopathological diagnosis of WM. Their median IgM was 2,980 (180–12,400) mg/dl, median BM involvement was 35% (5%–95%) and median B2M was 2.7 (1.4–13.7 mg/dL). Fifty-six (63%) and 66 (74%) of these patients demonstrated IgG (<700 mg/dL) and IgA (<70 mg/dL) hypogammaglobulinemia, respectively. Importantly, no correlation was seen between serum IgG and IgA levels and BM disease involvement, suggesting no direct impact of disease involvement on the production of uninvolved immunoglobulins. Given these findings, we undertook extensive germline and somatic sequence studies to determine if mutations existed within the APRIL/BLYS receptor TACI, which commonly is mutated in patients with Common Variable Immunodeficiency Disorder (CVID). Six of 29 (21%) sequenced patients displayed variants within TACI, which were present in both tumor cells (n=6) and germline (n=5). Importantly, patients displaying variants in TACI demonstrated significantly lower serum IgG (280 vs. 676 mg/dL; p=0.0009) and IgA (20 vs. 41 mg/dL; p=0.0006) levels versus patients without TACI variants. Lastly, sequence analysis of APRIL, BLYS as well as the TACI downstream TRAF proteins have led to detection of numerous variants which may further account for hypogammaglobulinemia among patients with normal TACI expression. These data suggest that aberrations in TACI signaling may account for IgG and IgA hypogammaglobulinemia in WM, akin to patients with CVID. Moreover, mutations within TACI signaling may have pathological implications for WM since a 300 fold increased incidence of lymphoma has been observed among CVID patients. The impact of TACI mutations on pathogenesis of WM is currently under investigation in our laboratory.
The Prognosis of Karyotypic Subgroups in Pediatric Acute Myeloid Leukemia Depends on the Alterations of KIT, MLL and FLT3 Genes: A Report from Japanese Childhood AML Cooperative Study Group.
Authors: Shimada Akira , Taki Tomohiko , Tabuchi Ken , Hanada Ryoji , Tawa Akio , Tsuchida Masahiro , Horibe Keizo , Tsukimoto Ichiro , Hayashi Yasuhide
Published: October 2019

Karyotypic abnormalities are associated with a prognosis of acute myeloid leukemia (AML). However, there are some patients who had poor prognosis among patients with the same karyotypic abnormalities. Recently, it is revealed that alterations of the genes including tyrosine kinases, lead to poor prognosis in AML. Genetic alterations associated with a poor prognosis remain to be clarified in karytotypic subgroups of AML. We performed the mutation analysis of FLT3, MLL, KIT, RAS and NPM in 158 pediatric AML patients who were enrolled in Japanese Childhood AML Cooperative Treatment Protocol, AML99, including 33 with normal karyotype, 46 with t(8;21), 7 with inv(16), 20 with 11q23 translocations, 13 with t(15;17), 10 with Down syndrome (DS) FAB-M7 and 29 other karyotypic abnormalities. In the 33 patients with normal karyotype, 9 FLT3-internal tandem duplication (ITD), 2 FLT3-D835 mutation (D835Mt), 8 MLL-partial tandem duplication (PTD), 2 KIT, 2 NRAS and 3 KRAS mutations were identified. FLT3-ITD and MLL-PTD were associated with a poor prognosis. Notably, NPM gene mutation was not identified in these patients. In the 46 patients with t(8;21), 2 FLT3-ITD, 1 D835Mt, 4 MLL-PTD, 8 KIT, 4 NRAS and 5 KRAS mutations were found. KIT mutations were associated with a poor prognosis. In the 7 patients with inv(16), 2 FLT3-D835Mt and 1 KIT mutations were identified. Only one patient with KIT mutation relapsed. In the 20 patients with 11q23 translocations, 1 D835Mt and 5 MLL-PTD were identified. MLL-PTD was associated with a poor prognosis in patients with 11q23 translocations. In the 13 patients with t(15;17), 3 FLT3-ITD and 3 D835Mt were found. FLT3-ITD was not associated with a poor prognosis. In the 10 patients with DS-M7, KIT and KRAS mutations were found in each one patient, showing no prognostic significance. In the 29 patients with other karyotypic abnormalities, 6 FLT3-ITD, 2 D835Mt, 4 MLL-PTD, 0 KIT, 2 NRAS and 2 KRAS mutations were found. FLT3-ITD and MLL-PTD were associated with a poor prognosis. Fifteen patients (9.5%) who had alterations of 2 of these 4 genes showed a poor prognosis. RAS gene mutations were found in 26 (16.5%) of 158 patients, but were not associated with the prognosis. These results suggest that a new different therapeutic strategy for the patients with these gene alterations in each subgroup, for example KIT mutations in t(8;21)-patients and MLL-PTD in patients with 11q23 translocation, is needed to improve the prognosis of pediatric AML.
Genomic Tumour Profiling with High-Density Oligonucleotide SNP Array in Sézary Syndrome.
Authors: Russo Giandomenico , Narducci Maria Grazia , Citterich Mauro Helmer , Picchio Maria Cristina , Critofoletti Cristina , Tocco Valeria , Scala Enrico , Sampogna Francesca , Frontani Marina , Arcelli Diego , Lombardo Giuseppe , Baliva Giannandrea , Caprini Elisabetta
Published: October 2019

Sézary Syndrome (SS) is a rare and aggressive form of Cutaneous T-Cell Lymphoma (CTCL) characterised by a distinct metastatic pattern mainly involving blood and skin and whose etiology and molecular pathogenesis are still unclear. Conventional cytogenetics studies have shown that most SS patients have chromosome aberrations; however allelotyping studies and genome-wide surveys for chromosome imbalances in this tumour are still very limited (Mao X. et al. 2003 Genes Chromosome Cancer 36:250–260). High-density single nucleotide polymorphism (SNP) arrays allow high-resolution and genome-wide detection of both loss of heterozygosity (LOH) and copy number (CN) abnormality. Therefore we used Affymetrix 10K SNP mapping array containing 11,560 tiled SNPs to investigate genomic aberrations of 13 individuals affected by SS. Genotype calls and signal information were obtained using GeneChip Operating Software (GCOS 1.4) and GeneChip DNA Analysis Software (GTYPE4.0). SNP calls were exported to be analysed with DNA-chip Analyser (dChip v1.3+) genotyping software which allows the simultaneous measurement of DNA copy number changes and LOH events (Zhao X. et al. 2004 Cancer Res. 64:3060–71; Lin M et al. 2004 Bioinformatics 20:1233–40).

Our findings indicate that chromosomes 17p, 10/10q and 9 are most frequently affected by LOH events, while gains of CN were observed more often for chromosome 17q and 8/8q. Among our patients almost all individuals showing loss of the 17p arm have also gain of the 17q, suggesting the presence of the isochromosome 17q, a frequently reported abnormality in SS. In addition to this, we characterised the chromosome LOH pattern identifying seven regions of significant loss shared by multiple tumours. Sample clustering based on significant LOH regions identified two groups of patients: one of them consists of 4 patients with a lower rate of chromosomal losses while the other contains 9 patients mainly characterised by the co-occurrence of LOH at chromosome 17 and chromosome 10.

The frequency and pattern of chromosomal changes in our group of 13 SS patients are in substantial agreement with previously described results using more conventional techniques, demonstrating the feasibility of the 10K SNP mapping array system to assess allelic imbalance in this tumour. The genome-wide approach and SNP high density allowed the identification of a larger number of LOH regions, including, however, those already described (chromosome 9p, 10q and 17p). Even though no significant statistical association can be observed due to the low number of cases available, we observed a lower overall survival in the group of 9 patients showing simultaneous LOH events at chromosome 17 and 10.
Real-Time Quantitative Polymerase Chain Reaction (RQ-PCR) Monitoring of Minimal Residual Disease (MRD) in Core Binding Factor Acute Myeloid Leukaemia (CBF AML).
Authors: Lane Steven W. , Saal Russell , Mollee Peter , Grigg Andrew , Taylor Kerry , Seymour John Francis , Kennedy Glen , Williams Bronwyn , Jones Mark , Grimmett Karen , Griffiths Vanessa , Gill Devinder , Hourigan Matthew , Marlton Paula
Published: October 2019

Background: CBF AML, with t(8;21)(q22;q22), inv(16)(p13q22) or t(16;16)(p13;q22) and the associated fusion proteins AML1/ETO or CBFβ/MYH11, has a favourable clinical prognosis although significant numbers of patients still relapse. We examined the prognostic utility of MRD monitoring by RQ-PCR and propose a simple model for prediction of impending haematological relapse.

Methods: Patients with CBF AML had samples collected at diagnosis, after induction and consolidation chemotherapy and at routine regular intervals thereafter. RQ-PCR, using the Applied Biosystems 7700 Sequence Detection System, was performed in triplicate and the final result was calculated by averaging 3 values, expressed relative to PGK levels. Stratified Cox regression was used to assess the impact of predictor variables (diagnostic, post-induction, post-consolidation RQ-PCR levels and presence of a 1 log10 increase in sequential RQ-PCR levels) on leukaemia-free survival (LFS).

Results: Of 46 patients identified with CBF AML, 29 had diagnostic, regular longitudinal samples and clinical follow up allowing further evaluation; 12 AML1-ETO and 17 CBFβ-MYH11. The median age was 39 years (range 7–68) with 52% male. Median follow up was 34 months (range 1–106) and median sample number was 6 (2–15). Twelve relapses occurred at a median of 11 months (range 4–17) from diagnosis. There were significant differences between transcript levels at diagnosis (median 1.9), post-induction (8.96*10−04), post-consolidation (5.01*10−05), in remission (1*10−06) or relapse (0.15) (p=0.01). Diagnostic, post-induction and post-consolidation RQ-PCR levels did not predict outcome. A log10 rise in a remission bone marrow sample correlated with adverse LFS and imminent risk of haematological relapse (HR 8.6). Relapses occurred a median of 60 days (range 45–272) after a log10 rise.

RQ-PCR levels Hazard Ratio (HR) LFS 95% CI P-value Diagnosis 0.22 0.02–2.9 0.25 Post-induction 1.3 0.8–2.1 0.36 Post-consolidation 0.8 0.5–1.2 0.27 1 log10 increase 8.6 1.8–42 0.008

Conclusions: A 1 log10 rise in transcript levels was a highly significant predictor of haematological relapse. Transcript levels at early post-treatment time points did not predict long term outcome, cautioning against de-escalation protocols based on these results. Prospective identification of high risk patients will enable clinical trials to address the efficacy of treatment initiated at molecular progression.
Minimal Residual Disease (MRD) Monitoring in CBFB-MYH11 Acute Myeloid Leukemia (AML) Is of Prognostic Relevance for Relapse-Free Survival.
Authors: Corbacioglu Andrea , Scholl Claudia , Eiwen Karina , Bullinger Lars , Frohling Stefan , Dohner Hartmut , Schlenk Richard F. , Dohner Konstanze
Published: October 2019

Detection of minimal residual disease (MRD) in acute myeloid leukemia (AML) associated with specific gene fusions is an important tool for the assessment of response to treatment and the individual risk of relapse. The real-time quantitative RT-PCR (RQ-PCR) method allows the quantification of fusion transcript levels at distinct time points during treatment. While in acute promyelocytic leukemia (APL) MRD monitoring has been clearly shown to be predictive for clinical outcome, the prognostic value of MRD in CBFB-MYH11 AML could not consistently been demonstrated yet. Small patient populations and the availability of bone marrow (BM)/peripheral blood (PB) samples at defined time points mainly hamper most studies. We evaluated the prognostic impact of MRD in a large cohort of CBFB-MYH11 AML by RQ-PCR. A total of 44 patients (16–60 years) were treated within one of the AMLSG treatment trials (AMLHD93 n=4, AMLHD98A n=27, AMLSG07-04 n=13). Patient samples (BM and/or PB) were collected at study entry (n=75), during treatment (n=199), and during follow up (n=140). Following high-dose cytarabine (HiDAC) consolidation therapy, patients received a second course of HiDAC (n=25); autologous stem cell transplantation (SCT) (n=13) or allogeneic SCT from a matched related family donor (n=6) depending on the treatment protocol. Median follow up was 22.5 months. Quantitative CBFB-MYH11 fusion transcript expression was measured by RQ-PCR using TaqMan technology. Primers and probes were chosen according to Europe Against Cancer (EAC) standard protocols. Sensitivities ranged from 10−3 to 10−4.Transcript levels at diagnosis ranged from 6208 to 312987 (median 34293.5). There was no prognostic impact of pretreatment transcript levels on relapse free survival (RFS). The ratio of transcript levels after 2 induction cycles and pretreatment levels ranged from 0 to 0.0049; again, this ratio had no impact on RFS. In contrast, during consolidation therapy 63% of the patients became RQ-PCR negative and RFS was significantly superior (RFS after 2 years 75%) compared to RQ-PCR positive patients (RFS after 2 years 32%) (p=0.03). After consolidation, seven of the RQ-PCR negative patients became positive at least in one BM-sample during follow up. Four patients developed transcript levels above 10 and all relapsed, whereas the three patients with transcript levels remaining below 10 are in continuous remission (p=0.0001). In our study, transcript levels during and after consolidation therapy are significantly associated with clinical outcome in CBFB-MYH11 AML. Risk-adapted therapy may be considered for those patients remaining positive during consolidation therapy. The identification of transcript levels above 10 after consolidation therapy might allow early treatment decisions.
Efficacy of an Antagonistic Anti-CD40 Monoclonal Antibody, HCD122 (CHIR-12.12), in Preclinical Models of Human Non-Hodgkin’s Lymphoma and Hodgkin’s Disease.
Authors: Long Li , Patawaran Montesa , Tong Xia , Kantak Seema , Aukerman Sharon L. , Pryer Nancy , Luqman Mohammad
Published: October 2019

Non-Hodgkin’s lymphoma (NHL) and Hodgkin’s disease (HD) account for about 9% of new cancer cases annually or 64,000 cases per year in the United States. Although the survival rate has significantly improved recently due to new combination therapy regimens, an unmet medical need remains for refractory or resistant patients. HCD122 is a fully human antagonistic anti-CD40 therapeutic monoclonal antibody (mAb) with a dual mechanism of action: blocking CD40 and CD40 ligand (CD40L) interactions and mediating antibody-dependent cellular cytotoxicity (ADCC). CD40 is expressed in all human B-cell malignancies, and the CD40/CD40L interaction is important for tumor cell proliferation and survival. Previously HCD122 was shown to potently inhibit CD40L-induced human B-cell and follicular NHL cell proliferation, mediate ADCC against CD40-positive human malignant B-cell lines and inhibit tumor growth in Burkitt’s lymphoma and multiple myeloma xenograft models. In this study the antitumor activity of HCD122 was assessed in preclinical models of HD and other subtypes of human NHL, such as Mantle cell and Follicular lymphoma. CD40 was expressed in 5 of 7 established human HD and 11 of 12 NHL tumor cell lines tested, including Hs445, HDLM-2, KM-H2, L428, L1236, Jeko-1 and WSU-NHL. Using purified human NK cells as effector cells, HCD122 mediated potent ADCC against these cell lines in vitro with a picomolar EC50. When human macrophages were used as effector cells, HCD122 also induced antibody-dependent cellular phagocytosis (ADCP) against the NHL Daudi cell line and the HD cell line Hs445. The antitumor activity of HCD122 was further evaluated in vivo in EBV-negative NHL and HD xenograft models. When tested in a staged human Mantle cell lymphoma Jeko-1 s.c. xenograft model in which treatment was initiated when the mean tumor volume reached 100 mm3, HCD122 was highly efficacious and induced complete tumor regression in 70% (7/10) of treated animals when administered intraperitoneally at 1 mg/kg weekly for 4 weeks. In a staged human HD L428 s.c. xenograft model, which expresses CD20 as well as CD40, the antitumor activity of HCD122 was compared to rituximab. HCD122 was highly efficacious and induced a mean 74 % tumor growth inhibition (TGI) when administered at 0.1 mg/kg weekly for 3 weeks (p<0.001). At the same dose and schedule, rituximab achieved only 40% TGI (HCD122 vs. rituximab: p<0.001). These data combined with our previous studies in multiple myeloma and EBV-positive Burkitt’s lymphoma models show that HCD122 is a potent anti-CD40 antibody with pronounced antitumor activity in both EBV-positive and EBV-negative malignant B cell preclinical models. HCD122 is currently in Phase I clinical trials in B-cell malignancies.
Improved Methodology for Detection of Mutations in the BCR-ABL Fusion Gene That Cause Resistance to the Tyrosine Kinase Inhibitor Imatinib.
Authors: Rothberg Paul G. , Vargas Roberto L. , Liesveld Jane L. , Ifthikharuddin Jainulabdeen J. , Frazier Sharon D. , Polochock Susan L.
Published: October 2019

Chronic myelogenous leukemia (CML) is caused by a balanced translocation between chromosomes 9 and 22 that creates the BCR-ABL fusion gene (FG) and activates the ABL tyrosine kinase (TK). Imatinib inhibits this TK and causes long-term remission in most CML patients. Relapse during imatinib therapy is often associated with mutations in the BCR-ABL FG that cause resistance to inhibition by imatinib. The usual procedure for detecting imatinib resistance mutations involves reverse transcription, PCR of the BCR-ABL FG, then nested PCR of the ABL TK to generate a template for sequencing. This procedure is suboptimal in a clinical laboratory because of the risk of carryover contamination, and extra time and workload, all due to the use of nested PCR. We describe a procedure for preparing sequencing template from the BCR-ABL FG that does not require nested PCR. The initial PCR employs a mixture of thermostable DNA polymerases that increases the sensitivity of the reaction for limiting concentrations of the desired target. One round of PCR provides enough template for sequencing. The PCR products are sequenced directly without gel purification, using three internal primers plus the downstream PCR primer as sequencing primers. The internal primers provide additional specificity resulting in a high quality sequence even if the PCR produces undesired products. Using this methodology we analyzed a series of eleven CML patient specimens selected because of a four-fold or greater increase in expression of the BCR-ABL FG during treatment with imatinib. The expression of BCR-ABL RNA was measured using a reverse transcription Q-PCR assay. The leftover cDNA from the expression assay was used as template for the PCR to generate the sequencing template. Five patients lacked a detectable mutation, and six had a mutation in the BCR-ABL TK. The six mutations found, G279E, F317L, L248V, M351T, F359C, and H396R, had all been seen previously and associated with resistance to imatinib. We conclude that detection of imatinib resistance mutations in the BCR-ABL FG is feasible without using nested PCR, although with a reduced analytical sensitivity. Elimination of the nested PCR step makes the procedure easier, quicker and less likely to suffer from carryover contamination. This method is a viable alternative for the detection of imatinib resistance mutations in the clinical laboratory.
Prognostic Value of Immunophenotype Analysis in Patients with Acute Promyelocytic Leukemia (APL) Treated with All-Transretinoic Acid and Anthracyclin Monochemotherapy.
Authors: Montesinos Pau , Rivas Concepcion , Rayon Consuelo , Vellenga Edo , de la Serna Javier , Bergua Juan , Parody Ricardo , Brunet Salud , Milone Gustavo , Sanz Miguel
Published: October 2019

Introduction: The prognostic significance of the expression pattern of certain cell surface markers in APL is controversial. Objectives: Analyse the impact of the expression of certain cell surface markers on complete remission rate (CR), overall survival (OS) and relapse free survival (RFS) in patients with APL included in multicenter trials PETHEMA LPA96 y LPA99.

Material and methods: Between 1996 and 2005, 734 patients were included in these 2 consecutive trials. Induction therapy consisted of ATRA and idarubicin, followed by three consolidation courses of anthracycline monochemotherapy with or without ATRA and followed by maintenance. Bone marrow immunophenotype analysis was performed at local or reference laboratories. Positivity was defined as more than 20% blasts expressing a specific antigen for the following antigens: CD34 (527 patients), CD33 (521), CD15 (520), CD13 (513), HLA-DR (495), CD2 (443), CD19 (433), CD7 (403), CD117 (395), CD56 (392), y CD11b (335). We performed univariate analysis to establish the impact of antigen positivity on CR rate, OS and RFS. Significant values were included in the multivariate analysis.

Results: A total of 664 patients (90%) achieved CR. The following variables were associated with decreased CR rate: WBC > 10x109/L, serum level creatinine > 1.4 mg/dl, age > 60 years, ECOG > 1, M3v and male gender. None of the cell surface antigens were significantly associated with CR rate. WBC, creatinine, age and gender were found to be independent prognostic factors for CR. Median follow up was 55 months. OS at 8 years was inferior in those patients with WBC > 10x109/L (67% vs 85%, p < 0.01), M3v (70% vs 83%, p < 0.01), age > 60 (56% vs 86%, p < 0.01), male gender (78% vs 83%, p=0.03), LPA96 trial (74% vs 84%, p=0.01) and CD2+ (76% vs 84%, p=0.04). Age, WBC and gender were independent factors for OS. RFS was inferior in those patients with WBC > 10x109/L (69% vs 93%, p < 0.01), high vs intermediate vs low risk (69% vs 91% vs 95%, p < 0.01), M3v (76% vs 88%, p < 0.01), BCR2 vs BCR3 vs BCR1 transcript (71% vs 81% vs 89%, p < 0.01), male gender (83% vs 90%, p=0.03), LPA96 trial (82% vs 87%, p=0.02) and CD2+ (75% vs 91%, p < 0.01). The risk of relapse category was the only independent factor for RFS. CD2+ APL (115/443 patients) was significantly associated with WBC > 10x109/L, M3v, BCR3, CD34+, CD56+, CD7+, and HLA-DR negative.

Conclusion: Of all the cell surface antigens analysed, only expression of CD2 was associated with an lower OS and RFS, due to its association with WBC > 10x109/L. In patients taking part in PETHEMA trials, immunophenotype analysis at presentation does not give additional prognostic impact from the previously established risk factors.
Relapse-Risk Stratification in Acute Promyelocytic Leukemia Patients by PML-RARa Transcript Quantification.
Authors: Santamaría Carlos , Chillón María C. , Fernández Carina , Martín-Jiménez Patricia , Alcoceba Miguel , Sarasquete María E. , Balanzategui Ana , García-Sanz Ramón , Miguel J.F. San , González Marcos
Published: October 2019

The detection of PML-RARa by real-time PCR (RQ-PCR) is becoming an important tool for monitoring minimal residual disease (MRD) in acute promyelocytic leukemia (APL) patients; however, the clinical value of using a RQ-PCR approach remains to be determined. Our aim was to analyze the correlation between relapse risk and MRD levels assessed by RQ-PCR at different phases of treatment, and to compare these data with conventional qualitative RT-PCR. Follow-up samples from 145 APL patients treated with the PETHEMA protocols were analyzed by RQ-PCR protocol from Europe Against Cancer program and by RT-PCR method from European BIOMED-1 Concerted Action. After induction therapy, no association was found between the presence of a RQ-PCR positive result and relapse. After the third consolidation course, three cases remained positive by RQ-PCR assays and two of them (67%) relapsed. By contrast, only 16 out of 119 (13%) patients with negative RQ-PCR relapsed. During maintenance therapy and out-of treatment, a significant correlation between a RQ-PCR positive result and a poor relapse-free survival was observed (p < 0.0001). All patients with > 10 PML-RARa normalized number copies (NCN) at maintenance or out-of therapy relapsed (n=19), while all patients with < 1 NCN within out-of treatment group remained in hematological remission (n=75). 8 patients with < 1 NCN during maintenance therapy finally relapsed, but all of them appeared beyond the end of treatment. In addition, in the intermediate group of patients (NCN 1–10) (n=18) relapse probability of 40% at 5 years was observed. All hematological relapses were predicted if a follow-up sample had been obtained within the previous 4 months. Based on the information provided by RQ-PCR in samples obtained after the end of consolidation and subsequently, a relapse risk stratification could be established for APL patients.
The Role of the Transcription Factor NFAT in the Pathogenesis of Non-Hodgkin’s Lymphomas.
Authors: Mueller Martin R. , Sasaki Yoshiteru , Sharma Sonia , Rajewsky Klaus , Rao Anjana
Published: October 2019

NFAT is a family of highly phosphorylated proteins residing in the cytoplasm of resting cells. Upon dephosphorylation by the Ca2+/calmodulin-dependent serine phosphatase calcineurin, NFAT translocates to the nucleus, where it induces the transcription of a large number of genes necessary for a productive immune response. NFAT signalling has also been implicated in lymphocyte homeostasis and its deregulation has been suggested to be involved in the pathogenesis of different malignancies. A recent immunohistochemical evaluation of approximately 300 Non-Hodgkin’s Lymphoma biopsy samples showed overexpression of NFAT2 in the majority of specimens with strong nuclear translocation in certain histologic subtypes (DLBCL, Burkitt’s Lymphoma) presumably reflecting activation of the NFAT pathway as part of their pathogenesis. Other recent studies have shown that NFAT activation leads to increased expression of different cell survival factors (CD154, BLyS) in several lymphoma subtypes (DLBCL, MCL).

To study the role of constitutive activation of the NFAT pathway on lymphomagenesis, we generated several hyperactivable NFAT mutants which will be used to generate transgenic mice expressing the mutant proteins from the ROSA26 locus. Here, we present the in vitro characterization of these hyperactivable NFAT proteins in cell lines and primary lymphocytes. The major docking site for calcineurin is located at the N terminus of the NFAT regulatory domain and has the consensus sequence PxIxIT (e.g. SPRIEIT in NFAT1 and NFAT2). Substitution of the SPRIEIT sequence of NFAT with HPVIVIT, a higher-affinity version obtained by peptide selection, increases the basal calcineurin sensitivity of the protein significantly. Similarly, a major kinase for NFAT is CK1, and mutation of the CK1 docking site (FSILF to ASILA in NFAT1, FDFEF to ADAEA in NFAT2) also leads to partial nuclear localization of NFAT proteins by decreasing rephosphorylation and nuclear export. In the current analysis we compared the different hyperactivable NFAT proteins with respect to dephosphorylation status and nuclear translocation using western blotting and immunocytochemistry. Whereas the wildtype NFAT protein was entirely localized in the cytoplasm and completely phosphorylated under resting conditions, we observed an increasing degree of nuclear translocation and dephosphorylation for the different mutant proteins (ASILA 20%, VIVIT 30–40%, ASILA-VIVIT 50–70%). This system will allow us to study the impact of different levels of NFAT activation on the pathogenesis of lymphomas in vivo.
Targeting xIAP Is More Important Than Bcl-Xl in Lymphomas with Constitutive Activation of NF-kB.
Authors: He Yanjuan , Cain Joan , Ratner Lee , Bernal-Mizrachi Leon
Published: October 2019

Pathways resulting in resistance to apoptosis are essential to the process of lymphomagenesis. One such pathway, the nuclear factor-kB (NFkB), has been shown to be a key element in coordinating the anti-apoptotic effect of these malignancies. However the mechanisms used by which NFkB prevents apoptosis are not well understood. It has been suggested that NFkB inhibits activation of the intrinsic, extrinsic and common apoptotic pathways. Previous work in our lab using two different virally mediated lymphoma models (Tax/HTLV1 and LMP1/EBV driven tumors) has identified two candidates that could explain these results: X chromosome-linked inhibitor of apoptosis (xIAP) and BCL-xL. Although the current literature extensively demonstrates the role of BCL-xL in lymphomas, little is known about the importance of xIAP in these malignancies. To answer this question we tested the apoptotic effect of etoposide or tumor necrosis factor (TNF) after knocking down bcl-xL and xIAP expression in our lymphoma models (SC and Daudi cell lines) using a lentivirus expressing siRNAs. After 24 hours of treatment with etoposide and TNF, we measured apoptosis by flow cytometry using double staining with Annexin V-Alexa Fluorescense and propidium iodide. Interestingly, xIAP siRNA-expressing cell lines demonstrated 2–4 fold increase in the induction of apoptosis after treatment with etoposide as compared to a nearly 2 fold increase in those expressing Bcl-xL siRNA (see Table below). No synergism was seen after treatment with TNF. Based on this finding, we then tested a novel small molecule, homolog smac, (SHC, kindly provided by Dr. PG Harren) to determine the possible therapeutic effect of xIAP inhibitors. After titration, the two most effective doses were selected (25 μM and 50 μM) to treat Daudi cell lines for 24hrs, with either etoposide or TNF. At doses of 25 μM , we observed a 2 fold increase in the induction of apoptosis produced by etoposide compared to that seen in control (DMSO + etoposide) or SHC alone and no synergism with TNF confirming the siRNA data. More importantly, at doses of 50 μM, SHC alone demonstrated activity with a 5 fold increase in apoptosis and a nearly 10 fold increase as compared to control (DMSO) when etoposide was added. Overall, we have demonstrated that xIAP and bcl-xL are important in mediating NFkB-resistance to apoptosis. However, our findings suggested that xIAP is a more potent anti-apoptotic signal and opens the door for further drug development aimed at testing xIAP-inhibitors in lymphomas.

Induction of Apoptosis in xIAP or Bcl-xL siRNA expressing cell lines siRNA/Compound Etoposide TNF Untreated xIAP 43.1 ± 17.6 17.04 ± 1.4 14.3 ± 2 SC Bcl-xL 18.39± 3.7 9.4 ± 0.22 12.5 ± 2.7 Luc/DMSO 14.9 ± 1.8 14.4 ± 5.6 14.03 ± 1.25 xIAP 9.2 ± 3.2 4.7 ± 0.48 4.6 ± 0.44 Bcl-xL 8.9 ± 0.5 5.3 ± 1.7 4.16 ± 0.4 Daudi Luc/DMSO 5.49 ± 1.71 4.28 ± 0.5 6.2 ± 0.9 SHC 25 μM 20.07 ± 4.8 12.8 ± 3.9 12.1 ± 3.2 SHC 50 μM 47.7 ± 14.55 38.3 ± 0.99 32.7 ± 8.99
Detection of Relapse in Diffuse Large B-Cell Lymphoma (DLBCL) and Hodgkin’s Lymphoma (HL): Observations and Implications for Post-Remission Radiologic Surveillance.
Authors: Xavier Marin F. , Schuster Stephen J. , Andreadis Charalambos , Downs Lisa , Diccion Bernadette , Nasta Sunita Dwivedy
Published: October 2019

According to reports in the literature since 1991, 5–30% of relapses of DLBCL and HL were identified by surveillance X-rays and CT scans in the post-remission setting. Recently, a large series of DLBCL relapses were analyzed and suggested that surveillance imaging can identify early relapse in a population of patients with a more favorable outcome (Leidtke et al, Ann Oncol 17: 909–913, 2006). We retrospectively evaluated 40 patients with relapses of HL and DLBCL after complete remission from three referral lymphoma practices between 2000–2006 at the University of Pennsylvania. Relapsed disease was identified after primary therapy or after salvage chemotherapy and high-dose therapy with autologous stem cell transplantation. These relapsed patients underwent post-remission surveillance CT scans with or without PET scans roughly every 3–4 months for the first 1–2 years and every 6–12 months thereafter for at least 5 years. Seventeen patients had DLBCL and 23 had HL. Among relapses, 22 (55%) were detected with surveillance imaging and 18 (45%) were detected by clinical findings (10 complaints of lymphadenopathy, 2 with B-symptoms, 2 with both B-symptoms and lymphadenopathy, 3 with pain, 1 with cough, 1 with physician-detected lymphadenopathy, and none by laboratory results alone). Among the asymptomatic relapses detected by surveillance imaging, PET scanning detected 4 cases of DLBCL (36%) and 3 cases of HL (27%) relapses not identified by other techniques. In summary, compared to prior reports, our series reveals a much higher proportion of relapses detected by surveillance imaging in both diseases. We have adhered to a protocol similar to the NCCN 2006 guidelines; including CT scans as a part of follow-up evaluation. This schedule is more intensive than surveillance reported in prior studies, perhaps accounting for our higher rate of radiographic relapse detection. FDG-PET scans performed in combination with follow-up CT scans detected additional early relapses not seen by CT scanning alone. Prospective screening studies looking at the role of FDG-PET scans combined with CT scans to detect earlier relapses is warranted. Whether early detection of relapse provides a survival advantage should be evaluated in larger prospective studies in both HL and DLBCL.