Rituximab in B-Cell Hematologic Malignancies: A Review of 20 Years of Clinical Experience

<p><strong>Article full text</strong></p> <p><br> The full text of this article can be found <a href="https://link.springer.com/article/10.1007/s12325-017-0612-x"><b>here</b>.</a><br> <br> <strong>Provide enhanced digital features for this article</strong><br> If you are an author of this publication and would like to provide additional enhanced digital features for your article then please contact <u>[email protected]</u>.<br> <br> The journal offers a range of additional features designed to increase visibility and readership. All features will be thoroughly peer reviewed to ensure the content is of the highest scientific standard and all features are marked as ‘peer reviewed’ to ensure readers are aware that the content has been reviewed to the same level as the articles they are being presented alongside. Moreover, all sponsorship and disclosure information is included to provide complete transparency and adherence to good publication practices. This ensures that however the content is reached the reader has a full understanding of its origin. No fees are charged for hosting additional open access content.<br> <br> Other enhanced features include, but are not limited to:<br> • Slide decks<br> • Videos and animations<br> • Audio abstracts<br> • Audio slides<u></u></p

image_3.tif

<p>Multiple myeloma (MM) is a proliferation of tumoral plasma B cells that is still incurable. Natural killer (NK) cells can recognize and kill MM cells in vitro and can limit MM growth in vivo. Previous reports have shown that NK cell function is impaired during MM progression and suggested that treatment with immunomodulatory drugs (IMIDs) such as lenalidomide (LEN) could enhance it. However, the effects of IMIDs on NK cells have been tested mostly in vitro or in preclinical models and supporting evidence of their effect in vivo in patients is lacking. Here, we monitored NK cell activity in blood samples from 10 MM patients starting after frontline induction chemotherapy (CTX) consisting either of association of bortezomib–lenalidomide–dexamethasone (Velcade Revlimid Dexamethasone) or autologous stem-cell transplantation (SCT). We also monitored NK cell activity longitudinally each month during 1 year, after maintenance therapy with LEN. Following frontline chemotherapy, peripheral NK cells displayed a very immature phenotype and retained poor reactivity toward target cells ex vivo. Upon maintenance treatment with LEN, we observed a progressive normalization of NK cell maturation, likely caused by discontinuation of chemotherapy. However, LEN treatment neither activated NK cells nor improved their capacity to degranulate or to secrete IFN-γ or MIP1-β following stimulation with MHC-I-deficient or antibody-coated target cells. Upon LEN discontinuation, there was no reduction of NK cell effector function either. These results caution against the use of LEN as single therapy to improve NK cell activity in patients with cancer and call for more preclinical assessments of the potential of IMIDs in NK cell activation.</p

image_2.tif

<p>Multiple myeloma (MM) is a proliferation of tumoral plasma B cells that is still incurable. Natural killer (NK) cells can recognize and kill MM cells in vitro and can limit MM growth in vivo. Previous reports have shown that NK cell function is impaired during MM progression and suggested that treatment with immunomodulatory drugs (IMIDs) such as lenalidomide (LEN) could enhance it. However, the effects of IMIDs on NK cells have been tested mostly in vitro or in preclinical models and supporting evidence of their effect in vivo in patients is lacking. Here, we monitored NK cell activity in blood samples from 10 MM patients starting after frontline induction chemotherapy (CTX) consisting either of association of bortezomib–lenalidomide–dexamethasone (Velcade Revlimid Dexamethasone) or autologous stem-cell transplantation (SCT). We also monitored NK cell activity longitudinally each month during 1 year, after maintenance therapy with LEN. Following frontline chemotherapy, peripheral NK cells displayed a very immature phenotype and retained poor reactivity toward target cells ex vivo. Upon maintenance treatment with LEN, we observed a progressive normalization of NK cell maturation, likely caused by discontinuation of chemotherapy. However, LEN treatment neither activated NK cells nor improved their capacity to degranulate or to secrete IFN-γ or MIP1-β following stimulation with MHC-I-deficient or antibody-coated target cells. Upon LEN discontinuation, there was no reduction of NK cell effector function either. These results caution against the use of LEN as single therapy to improve NK cell activity in patients with cancer and call for more preclinical assessments of the potential of IMIDs in NK cell activation.</p

Subcellular localization of the GFP-tagged, Bfl-1/Bax-derived (poly)peptides.

<p>MEF (left panels) and MEF DKO (right panels) cells were co-transfected with mitoDsRed plasmid (encoding DsRed2 fused to the mitochondrial targeting sequence from subunit VIII of human cytochrome c oxidase) and the GFP-tagged constructs. Subcellular distribution was analyzed by confocal microscopy 24 h after transfection. Confocal images showing GFP (green) and MitoDsRed (red) fluorescence. The DNA staining dye Topro-3 (blue) was used to visualize the nuclei. In merged images, the yellow color shows the co-localization of GFP and MitoDsRed at mitochondria. Scale bar, 10 µm.</p

μ-calpain cleaves Bfl-1 at two major sites in its N-terminus and releases a large C-terminal fragment with cytotoxic activity.

<p>(A) GST-Bfl-1(1–151) was digested with recombinant µ-calpain <i>in vitro</i> and the fragments were separated by SDS-PAGE (left panel). Bands corresponding to cleaved products (arrows) were analyzed by mass spectrometry (MS). A higher concentration of recombinant GST-Bfl-1(1–151) was treated with µ-calpain, products were separated by SDS-PAGE and blotted to PVDF (right panel). A sub-band (asterisk) was excised and subjected to Edman degradation, which indicated that Bfl-1 had been N-terminally cleaved between residues F71 and N72. (B) Schematic representation of the wild type Bfl-1 protein showing the location of the identified μ-calpain cleavage sites (asterisks, upper sequence), of mutant Bfl-1 protein with a 6 aminoacids deletion surrounding the two cleaved residues (Bfl-1DD, middle sequence), and of mutant Bfl-1 in which the region overlapping the first cleavage site was swapped with a structurally homologous region in Bcl2L10 (Bfl-1SD, bottom sequence) (C) Confirmation of the two calpain cleavage sites identified in Bfl-1 using noncleavable mutants. 293T cell lysates expressing GFP-tagged Bfl-1 constructs were exogenously treated with μ-calpain. Lysates containing equal amount of GFP-tagged Bfl-1 proteins were separated by SDS-PAGE and analyzed by western blot with an anti-GFP antibody to detect the full length protein and N-terminal truncated fragments and with a polyclonal anti-Bfl-1 antibody to detect C-terminal truncated fragments. Upper and lower panels represent two independent experiments with different time of exposure. (D) BJAB cells were cultured with or without treatment with TNF/CHX in the presence or absence of the calpain inhibitor ALLN. Lysates were separated by SDS-PAGE and the presence of a cleaved fragment was assayed by western blot using an anti-Bfl-1 antibody. (E) Secondary structure of Bfl-1 in which the nine helices of the protein are represented by boxes along with the different BH domains (left panel).The two cleavage sites mapped in (A) are indicated. A comparison with the previously published µ-calpain cleavage site in Bax is also shown <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038620#pone.0038620-Wood1" target="_blank">[26]</a> (bottom sequence). 3D structure of Bfl-1 (2VM6) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038620#pone.0038620-Herman1" target="_blank">[31]</a> and position of the different cleaved sites (red circles) are indicated. The different Bcl-2 Homology domains are colored in yellow (putative BH4), red (BH3), green (BH1) and blue (BH2). (F) FACS assays of Annexin V staining in HT1080 cells. Chimeric GFP constructs encoding GFP alone, or fusions of GFP with full-length Bfl-1 or Bax or with the various membrane-active α-helices corresponding to the C-terminal part of Bfl-1 or Bax, i.e. α5, α6 (PFD, pore forming domain) and α9 (FE, final exon) are represented. The α-helical topology of Bax and Bfl-1 corresponds to the structures solved in aqueous environment <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038620#pone.0038620-Herman1" target="_blank">[31]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038620#pone.0038620-Suzuki1" target="_blank">[50]</a>. Transfected cells were stained for phosphatidylserine exposure using Cy3-conjugated Annexin V and the percentage of apoptotic GFP-expressing cells was determined by FACS 24 hours post transfection (right panel). Death of GFP-expressing and staurosporine (STS)-treated cells were also monitored as controls. Graphs shown are representative of three independent experiments.</p

The Bfl-1-α9 peptide induces mitochondrial permeabilization through a membrane-destabilizing mechanism.

<p>Bfl-1-α9 peptide was incubated for the indicated times with mitochondria at lower concentrations (0.5 µM and 2.5 µM, top panels) and previously used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038620#pone-0038620-g004" target="_blank">figure 4</a> (10 µM and 25 µM, bottom panels). The release of cytochrome c and the expression of MitoHsp70 were monitored as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038620#pone-0038620-g004" target="_blank">figure 4</a>, combined with the detection of the external membrane associated protein hexokinase 1 (HK1) and the matrix-contained protein MnSOD.</p

Bfl-1-derived peptides have different abilities to permeabilize the MOM of mitochondria isolated from cultured cells.

<p>Peptides were incubated at different concentrations (10 µM and 25 µM) with isolated mitochondria for the indicated times (5, 15, 30 and 60 min) and the release of cytochrome c was monitored by immunoblot (IB). MitoHsp70 (mHsp70) was used as control indicative of equal-loading and proper isolation of the pellet fraction containing mitochondria (Mito) in comparison to the supernatant fraction (SN). Cytochrome c release assays were performed using iBMKW2 (wild type) and iBMKD3 (double KO Bax/Bak) for all tested peptides. For Bfl-1-α5, wild type MEF and MEF DKO (Bax/Bak −/− double KO) cells were used in parallel.</p

High DNA Methyltransferase <em>DNMT3B</em> Levels: A Poor Prognostic Marker in Acute Myeloid Leukemia

<div><p>It has been recently shown that DNA methyl transferase overexpression is correlated with unfavourable prognosis in human malignancies while methylation deregulation remains a hallmark that defines acute myeloid leukemia (AML). The oncogenic transcription factor <em>EVI1</em> is involved in methylation deregulation and its overexpression plays a major role for predicting an adverse outcome. Moreover, the identification of DNMT3A mutations in AML patients has recently been described as a poor prognostic indicator. In order to clarify relationship between these key actors in methylation mechanisms and their potential impact on patient outcomes, we analysed 195 <em>de novo</em> AML patients for the expression of <em>DNMT3A</em>, <em>3B</em> (and its non-catalytic variant <em>3B_NC</em>) and their correlations with the outcome and the expression of other common prognostic genetic biomarkers (<em>EVI1, NPM1, FLT3ITD/TKD</em> and <em>MLL</em>) in adult AML. The overexpression of <em>DNMT3B/3B_NC</em> is (i) significantly correlated with a shorter overall survival, and (ii) inversely significantly correlated with event-free survival and <em>DNMT3A</em> expression level. Moreover, multivariate analysis showed that a high expression level of <em>DNMT3B/3B_NC</em> is statistically a significant independent poor prognostic indicator. This study represents the first report showing that the overexpression of <em>DNMT3B/3B_NC</em> is an independent predictor of poor survival in AML. Its quantification should be implemented to the genetic profile used to stratify patients for therapeutical strategies and should be useful to identify patients who may benefit from therapy based on demethylating agents.</p> </div

Low HOXA9 expression associates with better survival outcome.

<p>Kaplan Meier analysis of two groups with expression levels above or below the cut-off at the first quartile for HOXA9 expression. (A) Event free survival (EFS) and (B) Overall survival (OS) were assessed in 193 patients.</p

Characteristics of the patients.

<p>F: female, M: male, pos: positive cases, neg: negative cases; M0 to M7: according to the French-American-British (FAB) diagnosis; 8UC (unclassified AML); nucleophosmin mutations (<i>NPM1</i>), fms-like tyrosine kinase-3 internal tandem duplications and tyrosine kinase domain mutations (<i>FLT3ITD/TKD</i>), mixed-lineage leukemia gene partial tandem duplications and rearrangements (<i>MLL PTD/R</i>); K: karyotype; CK complex karyotype (more than 3 abnormalities), NC-AML: Normal Cytogenetic Karyotype acute myeloid leukemia. To establish normal cytogenetic at least 20 metaphase cells from diagnostic bone marrow had to be evaluated and the karyotype had to be found normal in each mitosis. N: number of cases; +: positive cases; <i>DNMT3Am</i>: stands for mutated <i>DNMT3A; DNMT3B_NC</i>: stands for non-catalytic <i>DNMT3B.</i></p