bluuebunny/crossref_metadata_2025 huggingface dataset:
Bcr-Abl Regulates IRES-Dependent Translation of Lymphoid Enhancer Factor-1 in Chronic Myelogenous Leukemia.
Authors: Jimenez Judy , Zhang Min , Waterman Marian L. , Ong Tiong
Published: October 2019

The constitutive tyrosine kinase activity of Bcr-Abl leads to aberrant expression of multiple genes by several mechanisms, including dysregulation of transcription. Recently though, increasing attention has been focused on the effect of Bcr-Abl in dysregulating translation. Our group has previously documented the effects of Bcr-Abl on key regulators of cap-dependent translation and the role that this mechanism plays in transformation (Ly et al, Cancer Research, 2003; Prabhu et al., Oncogene, in press). Here we describe a novel form of translational control by Bcr-Abl. Specifically, we show how Bcr-Abl regulates cap-independent translation of Lymphoid Enhancer Factor-1 (LEF-1) via a bona fide internal ribosome entry site (IRES) in the 5′ untranslated region (UTR) of LEF1. Lymphoid Enhancer Factor-1 (LEF-1), a transcription factor that mediates Wnt signals via interaction with β-catenin, is often expressed in cancers derived from aberrant Wnt signaling. Lately, it has been reported that LEF1 transcripts are elevated in CML. We examined LEF-1 expression in primary CML cells and cell lines (K562 and Ba/F3-Bcr-Abl) and show that LEF-1 protein is detected in all patient-derived cells. Treatment of these cells with the Bcr-Abl imatinib mesylate (imatinib) inhibits LEF-1 expression in imatinib-sensitive cancers, but not in cancers that exhibit clinical resistance even though such cancers express imatinib-sensitive Bcr-Abl. For those cancers that are sensitive, inhibition of Bcr-Abl has a partial effect on LEF1 mRNA levels, and a significant effect on LEF-1 protein levels. LEF-1 protein is produced via two IRESs in it’s 5′ UTR. IRES-driven translation of LEF-1 was highly sensitive to Bcr-Abl as treatment with imatinib reduced IRES activity 5 fold. Transfection of CML cells with dicistronic mRNAs suggests that Bcr-Abl stimulates LEF-1 protein production through steps in the nucleus and cytoplasm. We propose that, in addition to its strong effects on cap-dependent translation in CML, Bcr-Abl is an important regulator of alternative translation pathways. mRNAs that are translated via IRES-dependent mechanisms are particularly relevant to cancer since they encode proteins that regulate cell proliferation and survival. Together, these observations underscore the important role which dysregulated translation plays in transformation, and suggest novel approaches with which to counteract the transforming properties of Bcr-Abl.
Increase in Leukemic Stem Cells Is Associated with Acceleration of BCR-ABL-Induced CML in P-Selectin-Deficient Mice.
Authors: Kong Linghong , Hu Yiguo , Peng Cong , Wang Zhu , Duffy Ted , Goodrich Ami , Li Shaoguang
Published: October 2019

The BCR-ABL oncogene causes chronic myeloid leukemia (CML) and B-cell acute lymphoblastic leukemia (B-ALL) in human, and CML-like disease and lymphoid leukemia in mice. Recent studies show that cell adhesion molecule P-selectin is a potent negative regulator of human hematopoietic progenitors. Our previous work shows that lack of P-selectin accelerates the development of BCR-ABL-induced CML in mice (Shawn et al, Blood, 104[7]:2163-2171, 2004, PMID: 15213099). We have observed that BCR-ABL-expressing Lin-c-Kit+ Sca-1+ bone marrow (BM) cells function as leukemic stem cells (LSCs). We tested whether LSCs play a role in the acceleration of CML development. We found that the percentage of LSCs in BM of P-selectin−/− CML mice was significantly higher than those in wild type C57BL/6 (B6) CML mice; other BM populations such as CMP, GMP, and MEP had no significant differences between the two groups of CML mice, suggesting that LSCs are responsible for the acceleration of CML disease when P-selectin is deficient. We also analyzed BM of B6 and P-selectin−/− mice and the BM of B6 and P-selectin−/− mice transplanted with BM transfected with empty vector virus. We found that there was no significant difference in the percentage of the stem cell population between the two groups of mice. This suggests that the higher percentage of LSCs in P-selectin−/− CML mice is caused by BCR-ABL but not by transplantation procedure associated with the P-selectin deficiency. Taken together, our results demonstrate that in CML mice, increased LSC population in P-selectin-deficient CML mice is associated with the acceleration of CML development.
Outcomes by Cytogenetic and Molecular Response at 12 and 18 Months of Imatinib in Patients with Newly Diagnosed Chronic Myeloid Leukemia (CML) in Chronic Phase (CP) in the IRIS Trial.
Authors: Baccarani Michele , Guilhot Francois , Larson Richard A. , O’Brien Stephen G. , Druker Brian J. ,   on Behalf of the IRIS Study Group
Published: October 2019

Long term data is now available from the International Randomized Study of Interferon vs. STI571 (IRIS). We performed landmark analyses for pts on imatinib (IM) at 12 and 18 months (mos) (n=509 and n=480 respectively) and stratified for CCyR (Ph+ 0%), PCyR (Ph+ >0–35%) or No MCyR (Ph+ >35%) at both timepoints. Progression-free survival (PFS) included all progressions to accelerated phase or blast crisis during treatment with IM, whereas event-free survival (EFS) also included any loss of MCyR/CHR, increase in WBC and CML-unrelated deaths on treatment as events. Overall survival (OS) considered all deaths including those after discontinuation of IM and irrespective of whether pts went on to transplant.

Outcomes in IM pts by cytogenetic response at 12 and 18 mos Estimated long-term outcomes at 60 mos (%) CcyR CcyR EFS PFS OS by 30 mos (%) by 60 mos (%) 12 mos landmark CCyR N = 350 93 97 95 – – PCyR N = 86 78 93 90 57 64 No MCyR N = 73 61 81 80 21 36 18 mos landmark CCyR N = 358 96 99 97 – – PCyR N = 66 80 90 90 38 50 No MCyR N = 56 69 83 82 11 27

Although the level of cytogenetic response was predictive for long-term outcomes, PFS and OS were not statistically significantly different between 12-mos CCyR and PCyR. Note that 64% of PCyR pts and 36% of pts without MCyR at 12 mos subsequently achieved CCyR. Using the 18-mos landmark, 50% of PCyR pts and 27% of pts without MCyR achieved CCyR at a later time. Long-term outcomes were evaluated based on available BCR-ABL transcript levels in pts with CCyR. A 3-log reduction from standardized baseline value in untreated pts was defined as a major molecular response (MMR). No pts who achieved both CCyR and MMR at 12 or 18 mos progressed to AP/BC by 60 mos. Approximately 5% of pts with CCyR but no MMR at 12 mos (p=0.007) and only 2% of CCyR pts without MMR at 18 mos (p=0.11) subsequently progressed. Of approximately 25% of CCyR pts with available PCR analysis who did not achieve MMR at 18 mos, about half did achieve MMR at a later time and their estimated EFS rate at 60 mths was 91%.

At 60 mos, 69% of patients randomized to IM remained on treatment. Achievement of CCyR and MMR by 12 and 18 mos after start of IM in newly diagnosed CML-CP are predictive for favorable long-term outcomes. About 50% of pts in PCyR or CCyR at these time points eventually achieve CCyR and MMR, respectively, on continued IM treatment. The ability to identify patients who may be late responders should be further studied.
Adaptive Autocrine Secretion of the Granulocyte Macrophage Colony Stimulating Factor (GM-CSF) Mediates Imatinib- and Nilotinib-Resistance in BCR/ABL-Positive Progenitors Via JAK-2/STAT-5 Pathway Activation.
Authors: Burchert Andreas , Wang Ying , Brendel Cornelia , Erben Philipp , Manley Paul W. , Hochhaus Andreas , Cai Dali , Neubauer Andreas
Published: October 2019

Overcoming disease persistence in chronic myeloid leukemia (CML) patients is of considerable importance to the issue of potential cure. Here we asked whether autocrine signaling contributes to survival of BCR/ABL positive cells in presence of imatinib mesylate (IM, Gleevec™) and nilotinib (AMN107, NI), a novel more selective Abl inhibitor. Conditioned media (CM) of IM-resistant LAMA84-cell clones (R-CM) was found to substantially protect IM-naïve LAMA cells and primary CML progenitors from IM- or NI-induced cell death. This was due to an increased secretion of the granulocyte-macrophage colony stimulating factor (GM-CSF) which was identified as the causative factor mediating IM resistance in R-CM. GM-CSF elicited IM and NI drug resistance via a BCR/ABL-independent activation of the JAK-2/STAT-5 signaling pathway in GM-CSF-receptor alpha receptor (CD116) expressing cells, including primary CD34+/CD116+ GM-progenitors (GMP). In a cell based resistance induction assay, GM-CSF enabled the evolution of IM- and NI-resistant colonies. When analysing GM-CSF expression in over 120 patient samples of first diagnosis CML patients and resistant patient, elevated mRNA and protein levels of GM-CSF were detected exclusively in IM-resistant patient samples, suggesting a contribution of GM-CSF secretion for IM and NI resistance in vivo. Importantly, inhibition of JAK-2 with AG490 abrogated GM-CSF-mediated STAT-5 phosphorylation and NI resistance in vitro. Together, this suggests that adaptive autocrine secretion of GM-CSF and cytokines in general may be a novel IM and NI resistance mechanism, which may also contribute to CML-persistence. GM-CSF protects CML-progenitors via a BCR/ABL-independent activation of the anti-apoptotic JAK-2/STAT-5 pathway. Inhibition of JAK-2 overcomes GM-CSF-induced IM and NI progenitor cell resistance providing a rationale for the application of JAK-2 inhibitors to eradicate residual disease in CML.
In CML, Marrow Fibrosis Relapses or Emerges in 5 - 6 % of Patients Per Year Independently Indicating Imatinib Failure and Shortened Survival Time of Patients.
Authors: Buesche Guntram , Hehlmann Rudiger , Ganser Arnold , Hecker Hartmut , Hochhaus Andreas , Heimpel Hermann , Heinze Barbara , Schoch Claudia , Gadzicki Dorothea , Mueller Martin , von Neuhoff Nils , Schnittger Susanne , Schlegelberger Brigitte , Frye Bernd , Georgii Axel , Kreipe Hans
Published: October 2019

Imatinib may reverse bone marrow fibrosis (MF) in chronic myeloid leukemia (CML). The risk of emergence or relapse of MF and its prognostic relevance are not known as yet. 110 patients with Ph+ CML recruited in two trials and treated with 400 mg imatinib / day were examined for the risk of MF and its prognostic significance.

Imatinib effectively reversed pretreatment MF (P = 0.0002), and pretreatment fiber increase did not significantly reduce the probability of achieving a hematologic, cytogenetic and major molecular response. However, during a follow-up period of 4 years, small foci with fiber increase (FFI) and MF relapsed in 13 % (5 / 38) of patients, and among patients with an initial normal fiber content (n = 72), FFI emerged in 26 % with transformation to full-blown MF in 38 % of them. FFI occurred in 3 / 27 cases with a major molecular response. The risk of emergence or relapse of FFI amounted to 13.7 % and of full-blown MF to 5.9 % per year, and the risk of acceleration and death increased to 90 % two years after occurrence of MF. FFI could be predicted neither by the risk score (Sokal, Hasford) nor the hematologic, cytogenetic and molecular response, nor by clonal evolution during imatinib treatment. In multivariate analysis, emergence or relapse of FFI and MF turned out to be a significant independent predictor of imatinib failure (P = 0.0003) as well as of acceleration and death of patients (P < 0.0009).

CONCLUSION: Although imatinib reverses initial MF in CML, it does not guarantee against relapse or emergence of MF which affects ~ 6 % of patients per year and therefore, may become a major problem during long-term therapy of CML. Focal evolution of MF should be included in the definitions of imatinib failure and accelerated phase, and continuing imatinib treatment at the current dose is no longer appropriate for patients with emergence of FFI or MF during therapy - regardless of the degree of hematologic, cytogenetic and molecular response.
Robust Anti-Leukemia CD4+ and CD8+ T Cell Responses in CML Patients Treated with Imatinib Mesylate.
Authors: Chen Christiane I.-U. , Johannsen Alexandre , Witteles Wesley , Maecker Holden T. , Lee Peter P.
Published: October 2019

Imatinib mesylate, a selective inhibitor of the bcr/abl tyrosine kinase, has revolutionized the treatment of CML and is the first-line therapy for most patients. Most CML patients in chronic phase achieve hematologic remission under treatment with imatinib, with up to 70% also achieving cytogenetic remission. However, imatinib therapy is not curative, as patients who discontinue the drug invariably relapse. Thus, the need to find alternate, potentially curative therapies remains. Low levels of CD8+ T cell responses to certain HLA restricted peptides have been detected in CML patients after successful treatment. To determine, if CML patients in remission on imatinib develop and sustain anti-leukemia CD4+ or CD8+ T cell responses, blood samples from patients before and several time points after treatment were collected and analyzed. Pre-treatment samples were utilized as sources of autologous leukemic cells to detect anti-leukemia T cell responses in post-treatment remission samples. Autologous leukemic samples alone and remission samples alone served as controls. In 7 of the 14 patients investigated, significant IFN-γ responses (p<0.01) in ELISPOT assay were detectable when patients achieved cytogenetic remission, and peaked at 10–15 months (median 36 SFCs, range 26–71 SFCs), before they slowly decreased (up to 46 months post-treatment) to levels similar to those from leukemic samples alone and remission samples alone (6 SFCs, range 0–13 SFCs). These results correlated with T cell responses in cytokine flow cytometry (TNF-α 1.4–40%, IFN-γ 1.0–6.6%, p<0.05), and showed a predominance for CD4+ T cells. Furthermore, TNF-α and IFN-γ production (p<0.05) was confirmed in CD4+ and CD8+ T cell clones generated in a remission sample from one patient using multiplex cytokine assay: 6.43–37.90 pg/ml for TNF-α, 14.90–110.42 pg/ml for IFN-γ. CD8+ T cell clones (HLA-A0201+) were tested for reactivity against HLA-A0201 restricted peptides derived from leukemia associated antigens proteinase 3, Wilms tumor 1 and bcr/abl, as well as tumor associated antigens p53, p68, and human telomerase (hTERT) in IFN-γ ELISPOT assays. No IFN-γ responses were detected in the CD8+ T cell clones when stimulated with the peptides compared to stimulation with an irrelevant peptide (0–15 SFCs, p=0.55), suggesting that these clones react to as yet to be defined leukemia associated antigens. Together, these data show that CD4+ T cells play an important role in anti-leukemia immune responses in patients in remission (sustained over time) and might contribute to killing of leukemic cells, possibly via TNF-α. Banked autologous leukemic cells could be used for vaccination of CML patients in order to enhance anti-leukemia T cell responses and may, in combination with imatinib, lead to eradication of residual leukemic cells with a durable cure.
STAT3: A Direct HMGA1 Gene Target Important in Lymphoid Malignancy.
Authors: Resar Linda , Dhara Surajit , Sumter Takita Felder , Mukherjee Mita , Turkson James , Jove Rich , Elbahloul Ossama , Aplan Peter , Lin Ying-Wei , Bhattacharya Raka
Published: October 2019

While the oncogenic properties of the high mobility group A1 (HMGA1) gene are well established, the molecular pathways that mediate transformation by HMGA1 have not been clearly defined. HMGA1 is widely overexpressed in hematologic malignancies and other human cancers. Moreover, its overexpression portends a poor prognosis in some tumors. The HMGA1 gene encodes the HMGA1a and -A1b chromatin binding proteins. We previously showed that overexpression of HMGA1a or -A1b confers a transformed phenotype in cultured, human lymphoid cells. More recently, we developed transgenic mice overexpressing HMGA1a and all mice develop aggressive lymphoid malignancy. Because HMGA1 proteins function in regulating gene expression, we hypothesize that overexpression of HMGA1 leads to neoplastic transformation by altering expression of specific target genes involved in this process. To identify genes that are regulated by HMGA1 in transformation, we used microarray gene expression profile analysis in cells transfected to overexpress HMGA1a or -A1b. Ninety-seven genes were differentially regulated by ≥ 2-fold in cells overexpressing HMGA1a and 103 genes were differentially regulated by ≥ 2-fold in cells overexpressing HMGA1b. Fifty-five of these genes were common to both the HMGA1a and -A1b cells. From the list of genes up-regulated in cells overexpressing HMGA1a or A1b, the gene encoding the signaling molecule, signal transducer and activator of transcription or STAT3, was studied further because of its prominent role in malignancy. HMGA1 binds to the STAT3 promoter in human leukemia cells in vivo by chromatin immunoprecipitation experiments and enhances transcription of the STAT3 promoter in transfection experiments. Both total STAT3 and activated STAT3 proteins are increased in leukemia cells from HMGA1a transgenic mice compared to lymphoid cells from control mice. Moreover, activated STAT3 recapitulates the transforming activity of HMGA1a in cultured cells. Blocking STAT3 function preferentially induces apoptosis in leukemia cells from HMGA1a transgenic mice, but not in control cells. STAT3 mRNA is also increased in human acute lymphoid leukemia samples that overexpress HMGA1a, suggesting that this pathway is also involved in human lymphoid leukemia. Our results indicate that STAT3 is a direct HMGA1a target gene that is both necessary and sufficient for transformation induced by HMGA1a.
The Histone Deacetylase Inhibitor Vorinostat (SAHA) Induces Apoptosis and Cell Cycle Arrest in Hodgkin Lymphoma (HL) Cell Lines by Altering Several Survival Signaling Pathways and Synergizes with Doxorubicin, Gemcitabine and Bortezomib.
Authors: Georgakis Georgios V. , Yazbeck Victor Y. , Li Yang , Younes Anas
Published: October 2019

Several epigenetic changes have been recently implicated in the pathogenesis of several human malignancies, including HL. This pathway is currently explored for cancer therapy using hypomethylating agents and histone deacetylase (HDAC) inhibitors. The purpose of this study was to examine the potential clinical value for HDAC inhibition in HL. Therefore, we examined the single agent activity of vorinostat (suberoylanilide hydroxamic acid, SAHA) in a panel of three well defined HL-derived cell lines (HD-LM2, L-428, and KM-H2). Furthermore, we determined the molecular mechanisms of vorinostst activity in these cell lines. Vorinostat showed antiproliferative effect in all HL cell lines in a time- and dose-dependent manner, as determined with the MTS assay. The most sensitive cell line was the HD-LM2 with an IC50 of 2 micromolar, but antiproliferative activity was observed at concentrations between 0.1 and 0.5 micromolar. This antiproliferative effect was due to cell cycle arrest at the G2/M phase, as determined by Propidium Iodide DNA staining and FACS analysis. This effect was most prominent in the HD-LM2 cells, as the G2M fraction increased by 6 fold within 24 hours of incubation with 10 micromolar of vorinostat, and by 4 fold when 5 micromolar was used. Cell cycle arrest was followed with an increase in Annexin-V binding indicative of induction of apoptosis. This apoptotic effect was associated with activation of the caspase pathway, which resulted in caspase 3 and PARP cleavage. Furthermore, the antiproliferative activity was associated with de-phosphorylation of three important survival signals that are known to promote HL survival: STAT6, Akt and ERK. To determine the potential synergy of vorinostat with conventional chemotherapy and modern targeted small molecules, we treated HL cell lines with the combination of vorinostat and doxorubicin, gemcitabine, and the proteasome inhibitor bortezomib. In all the combinations SAHA significantly enhanced the effect of the other drugs, as determined with the MTS assay and the calculation of the Combination Index. Moreover, the combination of vorinostat and bortezomib arrested the cells in G2/M phase more efficiently and showed increased rate of apoptosis than each drug alone. In contrast, there was no added benefit when vorinostat was combined with the heat shock protein-90 inhibitor 17-AAG or with the mTOR inhibitor CCI-779. Our data indicate that HDAC inhibition by vorinostat may have a clinical value in HL, especially when combined with bortezomib or with chemotherapy.
The Significance of BAALC Expression and CEBPa Mutations as New Prognostic Factors in Pediatric Acute Myeloid Leukemia with Normal Karyotype.
Authors: Mizushima Yasuhiro , Adachi Souichi , Taki Tomohiko , Shimada Akira , Hayashi Yasuhide , Tsukimoto Ichiro , Yui Masahiro , Kamitsuji Yuri , Hiraumi Yoshimi , Matsubara Hiroshi , Watanabe Motonobu , Watanabe Ken-ichiro , Nakahata Tatsutoshi
Published: October 2019

Approximately half of children with acute myeloid leukemia (AML) have no chromosomal or genetic abnormalities that can predict outcome. To improve the prognostic stratification on this heterogeneous group of patients, novel markers need to be investigated. Recently, CEBPa (CCAAT/enhancer binding protein alpha) mutations have been described as a favorable prognostic factor and the high expression level of BAALC (brain and acute leukemia, cytoplasmic) was found to be a poor prognostic marker, in adult AML with normal karyotype. To explore their significance in childhood, we studied the prognostic impact of the high expression level of BAALC and CEBPa mutations in pediatric AML with normal karyotype. Samples were available from pediatric patients 3 months to 15 years of age, treated on Japanese Childhood AML Cooperative Study Group Protocol, AML99. BAALC expression was determined by comparative real-time RT-PCR assay in 29 patients. The relative BAALC expression was determined using the comparative cycle threshold method. The median value of healthy volunteers was used as cutoff. The mutational analysis of CEBPa was performed in 49 patients with RT-PCR followed by direct sequencing. For samples carrying mutations, additional subcloning analysis were carried out. 72.4% (21 of 29) of patients had high BAALC expression and 27.6% (8 of 29) had low. High BAALC expression was associated with the FAB subtypes M0, M1, and M2, whereas low BAALC expression correlated with M4 and M5. Overall survival and event-free survival was 52.3%; 49.5% (high BAALC expression group) and 75.0%; 62.5% (low BAALC expression group), respectively. CEBPa mutations were detected in four patients (8.2 %, 4 of 49; two each in M1 and M2). N-terminal frameshift mutations and inframe insertions in the basic-leucine zipper (bZIP) domain were detected. Novel mutations (212 insertion (ins) C, 214–224 deletion CCCCGCACGCG, 720 ins CGCACC, 1074 ins AGA, 1092 ins CAC) were identified. One patient had mutations occurred in both N-terminal part and bZIP domain. Moreover, above patients with CEBPa mutations had not cooperating mutations with FLT3-ITD (fms-like tyrosine kinase 3 internal tandem duplication) and maintain in complete remission for at least 21 months duration. In conclusion, BAALC expression and CEBPa mutations may be prognostic factors in pediatric AML with normal karyotype.
Improved Measurement of Minimal Residual Disease (MRD).
Authors: Morley Alexander A. , Latham Sue , Brisco Michael J. , Sykes Pamela J. , Kuss Bryone , Waters Keith
Published: October 2019

A new and improved method for measurement of minimal residual disease (MRD) was developed.

Method

the total repertoire of leukaemic rearrangements of the immunoglobulin heavy chain (IgH) gene.was determined by performing multiple parallel Q-PCRs in microplates to determine usage of individual V and J segments. This enabled detection and quantification of clones ranging in size from 100% to approximately 0.03% of the leukemic population. MRD measurement involved nested Q-PCR using the specific V and J primers and internal primers based on the sequence of the rearrangement of interest.

Following informed parental consent, 25 children with B-ALL were studied at the end of induction therapy. Under general anesthesia, 4 aspirations, 2 from each iliac spine, were performed and on each sample MRD was measured on 2 different days.

This enabled definition of the laboratory and sampling factors important in measurement of MRD.

Results

Repertoire analysis was very effective in identifying rearrangements of the IgH gene suitable for use as molecular markers, being superior to the commonly-used BIOMED-2 protocol for identification of both large and small clones. Two or more rearrangements marking large clones were detected in 20 of the 25 patients.

The median MRD level at the end of induction was 2.1 x 10−5. A level of > 10−3 was seen in 4 patients and < 10−7 in 4. Sensitivity of detection in a single sample was approximately 2 x 10−6, which is 1–2 orders of magnitude better than current techniques.

Figure Figure

The SD of measurement depended on the number of target rearrangements present in the sample, reflecting the Poisson statistical uncertainty inherent in measurement of small numbers of events. For > 50 rearrangements SD was 0.23 log units, but below this level the SD rose steeply. 50 targets in 1 μg of DNA corresponds to an MRD of approximately 3 x 10−4.

Figure Figure

There was significant sampling error. In 1 patient there was 1000-fold MRD difference between the 2 iliac spines.

Conclusions

We recommend that, for MRD measurement,

-an aspiration should be performed from each iliac spine, with each sample being quantified separately and an average obtained -each measurement should involve at least 10 μg of good-quality DNA

The MRD value so obtained should have sufficient accuracy, sensitivity and precision for clinical decisions based upon the value to be made with confidence.

The described methods for MRD measurement should also be applicable to monitoring of all B-lymphocyte neoplasms, in addition to ALL.
Notch1 Mutations in Adult T Lymphoblastic Lymphoma and T-ALL.
Authors: Baleydier Frederic , Simon Arnauld , Bergeron Julie , Millien Corinne , Beldjord Kheira , Lepretre Stephane , Dombret Herve , Asnafi Vahid , Macintyre Elizabeth A.
Published: October 2019

Notch1 is recognised to be involved in human T-ALL oncogenesis, initially by implication of its intracellular domain in the rare translocation t(7;9) and more recently with the description of mutations in NOTCH1 exons coding the heterodimerisation (HD) +/− PEST domains in 50% of T-ALL. Favorable prognosis impact of Notch1 mutations has been shown in pediatric patients. Little data is available regarding Notch mutations in adult T-ALL, and even less in adult T Lymphoblastic lymphoma (T-LBL). We have therefore searched for Notch1 mutations in adult T-ALL and T-LBL, all of which have undergone comparable, TCR and oncogene based classification (TLX1/3, SIL-TAL1, CALM-AF10, LMO1/2, TAL1 and LYL1). We searched for mutations in exons 26, 27 and 34 by direct sequencing in 46 T-ALL (median age 27,5y. range 16–54; 6 < 18y) and 32 T-LL (median 31y., range 16–70y.; 3 < 18y.). Notch1 mutations were identified in 22/46 (48%) of T-ALL and 16/32 (50%) of T-LBL. The type of mutations differed, insofar as HD only mutations were seen in 16/22 (73%) of mutated T-ALL but only 4/16 (25%) of mutated T-LL whereas PEST only mutations were seen in 2/22 (9%) T-ALL compared to 8/16 (50%) of T-LBL. The incidence of HD and PEST mutations was identical in T-ALL (4/16; 25%) and T-LBL (6/22; 27%). Combining T-ALL and T-LBL, TLX1hi or TLX3 expression was seen in 7/71 (10%) and 5/71 (7%) cases respectively. Notch1 mutations were seen in 7/7 TLX1+ cases but only 1/5 TLX3 (p=0.004). With respect to stage of maturation arrest, Notch mutations in T-ALL were relatively rare in mature TCR+ cases (2/15 vs. 20/31 p=0.001), whereas they were found at all stages of maturation arrest in T-LBL.

These data demonstrate that Notch 1 mutations are as frequent in T-LBL as T-ALL, but more frequently involve the PEST domain. They are in favour of preferential occurrence of NOTCH1 mutations in TLX1 relative to TLX3 proliferations, suggesting that evaluation of prognostic impact of Notch1 should not be performed independently of evaluation of TLX1/3 status, since TLX1 generally identifies good prognosis and TLX3 poor prognosis T-ALL. Within the 46 LALA94 adult T-ALLs, Notch1 mutated cases were of marginally superior prognosis (p=0.50); this is currently being evaluated within the ongoing adult GRAALL03 T-ALL trial.
FLT-3 Activity and Its Response to Drugs Can Be Determined in AML Blast Cells by FLT-3 Phosphorylation Status Using Flow Cytometry.
Authors: Grafone Tiziana , Palmisano Michela , Ottaviani Emanuela , Martelli Alberto Maria , Cappellini Alessandra , Follo Matilde , Testoni Nicoletta , Paolini Stefania , Baccarani Michele , Martinelli Giovanni
Published: October 2019

One of the most common molecular defects identified in acute myeloid leukemia (AML) patients is an activating mutation of FLT3 tyrosine kinase. The identification of activated FLT3 as a contributor to the cause and progression of much leukemia has led to its consideration as a potential target for therapy. Since small molecule FLT3 kinase inhibitors are actually in clinical trials; a robust and standardized method for screening of FLT3 receptor activation is necessary.

We evaluated the expression level of FLT3 receptor (CD135) by Facs analysis. We developed a flow cytometry method to measure FLT3 phosphorylation (P-FLT3) in samples with <10 (e)5 cells. The amount of P-FLT3 in the samples was determined as the mean fluorescence intensity (MFI). The P-FLT3 status of the treated samples was expressed as a percentage of the untreated control (100%). The method was first validated in FLT3 wild-type (HL-60) and mutant (MV4-11/ITD+) as well as FLT3 negative (K562) cell lines. The method also provides to be reproducible with samples AML from patients. Analysis was performed after exposure to drugs, in vitro and in vivo. In response to increasing drugs concentration (CEP-701 and SU11657) there was a linear reduction in P-FLT3. The results validate a rapid method to detect P-FLT3 protein at the single cell level by flow cytometry, and enable an accurate assessment of FLT3 kinase activity in blast cells in response to novel tyrosine kinase inhibitors.
Specific Tumor Targeting and Potent Bystander Killing with SGN-35, an Anti-CD30 Antibody Drug Conjugate.
Authors: Okeley Nicole M. , Alley Stephen C. , Cerveny Charles G. , Andreyka Jamie , Sanderson Russell J. , Anderson Martha , Benjamin Dennis R. , Law Che-Leung , Sievers Eric , Ihle Nathan C.
Published: October 2019

CD30 is expressed on the surface of Reed Sternberg cells in Hodgkin’s Lymphoma (HL) and on anaplastic large cell lymphoma (ALCL) cells. The anti-mitotic agent monomethyl auristatin E (MMAE) has been conjugated to cAC10, the anti-CD30 monoclonal antibody SGN-30, to yield the antibody-drug conjugate SGN-35. SGN-35 is internalized into lysosomes of CD30-positive cells and MMAE is released intracellularly when the protease-sensitive linker is cleaved by lysosomal proteases such as cathepsin B. In mouse xenograft models of HL and ALCL, complete remissions have been observed at well tolerated doses of SGN-35. To characterize further the mode of action in vivo, mice with established CD30-positive xenografts were treated with SGN-35, and the concentration of released MMAE was measured in the tumor and serum. The tumor concentrations of released MMAE achieved were several hundred-fold above the IC50 for MMAE in tumor cell cultures. Peak tumor to serum ratios for released MMAE reached more than 1000:1. In tumors and serum, the peak released MMAE concentrations were achieved 3 days and 1 hour post dose, respectively. As a considerable portion of the tumor volume in HL consists of a CD30-negative cellular infiltrate, the ability of SGN-35 to kill CD30-negative cells in mixed cell populations was investigated. First, cell growth medium from the SGN-35-treated HL cell line L540cy (CD30 positive) was shown to be cytotoxic to the SGN-35-insensitive Ramos cells (CD30 negative). Measurements with mass spectrometry confirmed that MMAE was released into the growth medium of SGN-35-treated L540cy cells, and that the observed concentration of MMAE was consistent with the cytotoxic effect on the Ramos cells. Second, a mixed culture of Karpas 299 cells (ALCL, CD30 positive) and Ramos cells was treated with SGN-35 and examined by FACS. The viability of both cell populations was reduced or eliminated, in contrast to proliferation observed in single Ramos cultures treated with SGN-35 or untreated mixed cultures. These results demonstrate that SGN-35 effectively targets the potent cytotoxic agent MMAE to CD30-positive tumor cells, while free MMAE released by these tumor cells is also available for killing CD30-negative infiltrating bystander cells. SGN-35 is a promising treatment for tumors with homogeneous (ALCL) and heterogeneous (HL) expression of CD30. Clinical trials with SGN-35 are expected to begin by the end of 2006.
Characterization of T-Cell Lymphoma Cell Lines with a Novel t(9;14)(q34;q11.2) Rearrangement: Does gamma-Secretase Inhibitor Sensitivity Depend on Intragenic NOTCH1 Breakpoint?.
Authors: Suzuki Shinsuke , Nagel Stefan , Schneider Bjoern , Kaufmann Maren , Anders Dorothea , Tsubouchi Hirohito , Uozumi Kimiharu , Arima Naomichi , Drexler Hans G. , MacLeod Roderick A.F.
Published: October 2019

Activating mutations and deletions affecting specific NOTCH1 protein domains have been recently shown to occur widely in T-cell neoplasia, e.g. in T-acute lymphoblastic leukemia (T-ALL). However, knowledge of NOTCH1 chromosomal alterations is largely based on a single cell line model (SUP-T1) with t(7;9)(q35;q34) in which NOTCH1 truncated at exon 24 is juxtaposed with TCRB. We describe the characterization of a novel rearrangement, t(9;14)(q34.3;q11) in two T-cell lymphoma cell lines, HD-MAR and HT-1. FISH analysis using fosmid clones and sequencing of fragments identified by long distance inverse PCR showed that in both cases t(9;14) effected tail-to-tail juxtaposition of intron 27 of NOTCH1 with TCRA genes, namely 5′-TRAV40 in HD-MAR, and intron 2 of TRAV5 in HT-1. Thus, in both cell lines t(9;14) places NOTCH1, truncated immediately 3′ of the HD-domain, under transcriptional control of TCRA. The 14q11.2 breakpoints in HD-MAR and HT-1 lie, respectively, near the proximal E-delta enhancer and amid a cryptic enhancer region represented by a cluster of T-cell specific DNase-I hypersensitive sites. Western blotting revealed prominent expression of truncated activated NOTCH1 polypeptides, ranging in size from 100 to 115 kDa in both cell lines. Antibodies recognizing ANK and TAD domains, believed essential for inducing T-ALL, detected the aberrant polypeptides. Moreover, treatment with gamma-secretase inhibitor (GSI) altered expression patterns of NOTCH1 polypeptides and induced growth inhibition due to G0/G1 cell cycle arrest in both t(9;14) cell lines, in stark contrast to GSI-resistant SUP-T1 cells wherein truncation occurs before the heterodimerization (HD) domain. (Another recently described t(7;9) cell line (CUTLL1) which is GSI-sensitive also carries a NOTCH1 breakpoint at intron 27.) The same protein species were not detectable by antibodies recognizing the transmembrane domain of NOTCH1 which requires GS for exposure suggesting nuclear access requires GS-cleavage. Immunostaining confirmed extranuclear blocking of NOTCH1 in response to GSI in HD-MAR/HT-1 but not in SUP-T1. In contrast, repression of HES1 occurred in response to GSI irrespective of NOTCH1 breakpoint location, suggesting its non-involvement in growth signaling. In addition to providing cell line models for a new NOTCH1 disease translocation, these data suggest that the sensitivities of T-cell neoplasias bearing NOTCH1 translocations may critically depend on whether 9q34 breakpoints lie upstream or downstream of the HD domain.
Gene Expression Profiling Comparison between the Major Mature B-Cell Neoplasms and Their Normal Cellular and Anatomic Counterparts: Identification of Candidate Genes Potentially Involved in Lymphomagenesis.
Authors: Blin Nicolas , Bossard Celine , Harousseau Jean-Luc , Charbonnel Catherine , Gouraud Wilfried , Campion Loic , Magrangeas Florence , Minvielle Stephane , Loiseau Herve Avet
Published: October 2019

Gene expression profiling has provided new insights into the understanding of mature B cell neoplasms by relating each one to its normal counterpart, so that they can be to some extent classified according to the corresponding normal B-cell stage. Thus, diffuse large B cell (DLBCL) and follicular lymphoma (FL) have been related to a germinal center precursor whereas mantle cell lymphoma (MCL) or marginal zone lymphoma (MZL) are more likely to derive from naïve and memory B cell, respectively. However, little is still known about the physiopathology of B-cell lymphomas and particularly the deregulated pathways involved in their oncogenesis. To further investigate that point, we performed laser capture microdissection (LCM) of the three anatomic lymphoid compartments (i.e germinal center, mantle zone and marginal zone) taken from nine normal spleens and lymph nodes and magnetic cell separation of the four normal B cell subpopulations (i.e naïve B cells, centroblasts, centrocytes and memory B cells) purified from twelve normal tonsils for gene expression profiling by cDNA microarray. These molecular profiles have been compared to those of the four most frequent mature B cell neoplasms in adult (i.e DLBCL, FL, MZL and MCL), each one isolated from five previously untreated patients. Unsupervised analysis by hierarchical clustering of the normal anatomic and cellular populations could discriminate the germinal from the extra-germinal populations by genes involved in cell proliferation (e.g. E2F5, CCNB2, BUB1B and AURKB), DNA repair (e.g. PCNA and EXO1), cytokine secretion (e.g. IL8, IL10RB, IL4R and TGFBI) and apoptosis (e.g. CASP8, CASP10, BCL2 and FAS). Supervised analysis of the comparison between each B-cell lymphoma and its anatomic and cellular physiologic equivalent identified molecular deregulations concerning several genes’families characterizing the different histologic subtypes. Genes associated with cellular adhesion and ubiquitin cycle were significantly up-regulated in MCL (FCGBP, ITGAE, USP7, VCAM1) and MZL (CTGF, CDH1, ITGAE) whereas germinal center derived lymphomas (i.e. DLBCL and FL) mainly showed up-regulation of genes involved in cell proliferation (TNFRSF17, SEPT8) and immune response (FCER1G, XBP1, IL1RN). Few deregulated genes were common to the four subtypes, principally associated with cell proliferation (CYR61, GPNMB), cytosqueleton organization (EPB41L3) and carbohydrates metabolism (GNPDA1), suggesting potential similar oncogenic pathways. Those preliminary results are compatible with both subtype-specific and overall mechanisms of lympomagenesis and should be verified in a wider range of samples to confirm the oncogenic events involved in this heterogeneous disease.
Alternative Splicing Generates a BCL6 Isoform Encoding a Compact Repressor.
Authors: Shen Yulei , Ge Baosheng , Ramachandrareddy Himabindu , McKeithan Timothy , Chan Wing-Chung
Published: October 2019

BCL6 encodes a BTB/POZ zinc finger (ZF) transcriptional repressor which is essential for normal germinal center (GC) reactions. Its dysregulated expression contributes to the pathogenesis of B cell non-Hodgkin lymphoma (NHL). The BCL6 gene consists of ten exons, BCL6 translation start site is located in exon 3, and the six-ZF DNA binding domain is encoded by exons 7 to 10. BCL6S, was cloned from the cDNA of a BCL-6 positive cell line, DHL-16. DNA sequencing showed that BCL6S was a normal splicing isoform that excludes the entire exon 7 of the BCL6 gene and encodes a 650 amino acid (aa) protein. BCL6S was detected by RT-PCR in all human cell lines and tissues expressing BCL6, but was not detected in the mouse B cell lymphoma cell line A20. BCL6S accounts for 1/8 to 1/10 of total BCL6 transcripts. Luciferase reporter assays demonstrated that BCL6S could repress typical BCL6 target genes ( BCL6, CD23b, Blimp1, MIP1α ) as effectively as the full length BCL6. BCL6S retains the last four ZFs of BCL6 and localizes in the cell nucleus. Protein-protein interaction assays confirmed that BCL6 and BCL6S could form homodimers and heterodimers. DNA binding assays showed no affinity differences between BCL6 and BCL6S; however, a single mutation at the N-terminus of the POZ domain (L19 to H) not only disrupted BCL6S dimerization but also significantly decreased the DNA binding affinity, indicating that BCL6 dimerization may stabilize the ZF-DNA binding. In conclusion, we have identified a novel BCL6 splicing isoform. BCL6S, a compact repressor as potent as BCL6. POZ dimerization plays an important role in stabilization of BCL6 DNA binding whereas the first two ZFs are not absolutely required. The biological role of BCL6S in GC B cells needs to be further investigated.
Primary Extranodal Follicular Lymphoma: Clinicobiological Features and Outcome.
Authors: Mercadal Santiago , Martinez-Pozo Antonio , Bosch Francesc , Gine Eva , Muntanola Ana , Salamero Olga , Gutierrez Gonzalo , Montoto Silvia , Colomo Luis , Campo Elias , Montserrat Emili , Lopez-Guillermo Armando
Published: October 2019

Follicular lymphoma (FL) is typically a nodal disease. Primary extranodal FLs, that represent less than 10% of the cases, might have differentiated clinicobiological features. The aim of the present study was to analyze the main clinicobiological characteristics, response to therapy and outcome of a series of patients with FL primarily extranodal in origin, and compare them with nodal FLs. Twenty two patients (12M/10F; median age, 59 years) with FL with primary origin in extranodal location diagnosed in a single institution during a 24-year period, with primary origin in extranodal location were the subject of the study. The control group was constituted by 212 patients with nodal FL diagnosed during the same period of time. Main clinicobiological features were recorded and analyzed. The sites of the primary disease were: skin, 5 cases; Waldeyer’s ring, 4; GI tract, 3; bone marrow, CNS and parotid (two cases each); and pancreas, thyroid, kidney and orbit (one case each). Main histological and clinical features are listed in the table. Treatment was given without considering the nodal or extranodal origin of the disease and consisted of: monotherapy with alkylating agents (38 cases), polychemotherapy (149), and fludarabine alone or with other drugs (14) and others, including surgery and observation (33). CR rate was higher in extranodal than in nodal FL (82% vs. 53%, respectively; p=0.02), but no differences were found in overall survival. FLIPI score was the most significant variable predicting overall survival in the global series as well as in either in nodal or extranodal FL.

In conclusion, extranodal FL had some peculiar clinicobiological features with respect to nodal cases. Regarding the outcome, although patients with extranodal FL showed a higher CR rate, the overall survival was similar in both groups.

Extranodal FL (N=22) Nodal FL (N=212) p Age (median, range) 59 (28–82) years 55 (24–93) years NS Sex (M/F) 12/10 100/112 NS Histological grade 3 (%) 12 10 NS CD10+(%) 85 90 NS Bcl2+ (%) 67 91 NS Bcl2/JH (%) 37 65 0.03 Stage IV (%) 50 64 NS Bone marrow + (%) 36 62 0.02 LDH > 450 IU/L (%) 15 24 NS B2-microglobulin > 2.3 mg/L (%) 7 41 0.058 High-risk FLIPI (%) 19 35 NS CR rate (%) 82 53 0.02 5-year OS (%) 75 74 NS 5-year FFS (%) 67 27 <0.02
Front-Line Consolidation with Autologous Stem Cell Transplantation in Patients with Nodal Aggressive Peripheral T-Cell Lymphoma (PTCL). A Prospective Study of the GEL-TAMO.
Authors: Rodriguez Jose , Conde Eulogio , Gutierrez Antonio , Arranz Reyes , Leon Angel , Marin Julian , Bendandi Maurizio , Albo Carmen , Caballero Maria Dolores
Published: October 2019

Background: Nodal aggressive PTCL have a poor prognosis with conventional chemotherapy regimens for aggressive non-Hodgkin lymphomas. Therefore innovative approaches are needed. Among them, strategies including consolidation with high-dose chemotherapy and autologous stem cell transplantation in front line therapy may improve the outcome of these diseases.

Aim: To report the experience of a national cooperative group in 26 patients with aggressive nodal PTCL who underwent treatment including ASCT as consolidation of front line therapy.

Patients and Methods: Twenty-six patients Gallium scan positive with nodal PTCL entered into the study: 11 cases of PTCL-unspecified (42%), 8 ALCL (31%) and 7 AIL (27%). Patients received three courses of MegaCHOP and subsequently were evaluated by CT scan and Gallium scan. Those patients Gallium scan negative received another course of MegaCHOP and then the transplant. Those patients Gallium scan positive after 3 courses of MegaCHOP received 2 courses of the IFE regimen (Ifosfamide 3.3g/m2 days 1–3 and Etoposide 300 mg/m2 days 1–3) and if at least achieved a partial response received the transplant otherwise they are out of the study. Median age was 44 years, 96% of the patients presented Ann Arbor stage III or IV; 54% presented B symptoms; 31% bone marrow involvement and 72% of the patients presented 2–3 factors of the a-IPI.

Results: Thirteen patients (50%) achieved a CR after 3 courses of MegaCHOP and 12 patients received IFE as salvage therapy. Twenty patients out of the 26 initial patients were able to proceed to the transplant. After the transplant 85% of patients achieved a CR. The 6 patients who did not received the transplant was due to progression of the disease in 4 cases, lethal toxicity in one case and refusal in another case. With a median follow up of 35 months for alive patients, the overall survival (OS) and progression-free survival (PFS) at 3 years were 73% and 53%, respectively. OS, PFS and disease-free survival for the 20 patients who underwent the ASCT consolidation was 84%, 53% and 63%, respectively.

Conclusion: Early salvage therapy for patients not in CR after 3 courses of chemotherapy and ASCT consolidation for chemosensitive patients may improve the outcome of patients with nodal aggressive PTCL. Larger series and longer follow up are needed to confirm this promising approach.