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Purpose: To determine the role of CD49d for response to Bruton's tyrosine kinase inhibitors (BTKi) in patients with chronic lymphocytic leukemia (CLL). Patients and Methods: In patients treated with acalabrutinib (n = 48), CD49d expression, VLA-4 integrin activation, and tumor transcriptomes of CLL cells were assessed. Clinical responses to BTKis were investigated in acalabrutinib- (n = 48; NCT02337829) and ibrutinib-treated (n = 73; NCT01500733) patients. Results: In patients treated with acalabrutinib, treatment-induced lymphocytosis was comparable for both subgroups but resolved more rapidly for CD49d+ cases. Acalabrutinib inhibited constitutive VLA-4 activation but was insufficient to block BCR and CXCR4–mediated inside–out activation. Transcriptomes of CD49d+ and CD49d− cases were compared using RNA sequencing at baseline and at 1 and 6 months on treatment. Gene set enrichment analysis revealed increased constitutive NF-κB and JAK–STAT signaling, enhanced survival, adhesion, and migratory capacity in CD49d+ over CD49d− CLL that was maintained during therapy. In the combined cohorts of 121 BTKi-treated patients, 48 (39.7%) progressed on treatment with BTK and/or PLCG2 mutations detected in 87% of CLL progressions. Consistent with a recent report, homogeneous and bimodal CD49d-positive cases (the latter having concurrent CD49d+ and CD49d− CLL subpopulations, irrespective of the traditional 30% cutoff value) had a shorter time to progression of 6.6 years, whereas 90% of cases homogenously CD49d− were estimated progression-free at 8 years (P = 0.0004). Conclusions: CD49d/VLA-4 emerges as a microenvironmental factor that contributes to BTKi resistance in CLL. The prognostic value of CD49d is improved by considering bimodal CD49d expression.

Supplementary Figure S4. Clinical impact of CD49d bimodal expression on durability of BTKi therapy.

Supplementary Table S6. DEGs for Baseline Comparison of CD49d+ and CD49d

Supplementary Methods

Purpose:To determine the role of CD49d for response to Bruton's tyrosine kinase inhibitors (BTKi) in patients with chronic lymphocytic leukemia (CLL).Patients and Methods:In patients treated with acalabrutinib (n = 48), CD49d expression, VLA-4 integrin activation, and tumor transcriptomes of CLL cells were assessed. Clinical responses to BTKis were investigated in acalabrutinib- (n = 48; NCT02337829) and ibrutinib-treated (n = 73; NCT01500733) patients.Results:In patients treated with acalabrutinib, treatment-induced lymphocytosis was comparable for both subgroups but resolved more rapidly for CD49d+ cases. Acalabrutinib inhibited constitutive VLA-4 activation but was insufficient to block BCR and CXCR4–mediated inside–out activation. Transcriptomes of CD49d+ and CD49d− cases were compared using RNA sequencing at baseline and at 1 and 6 months on treatment. Gene set enrichment analysis revealed increased constitutive NF-κB and JAK–STAT signaling, enhanced survival, adhesion, and migratory capacity in CD49d+ over CD49d− CLL that was maintained during therapy. In the combined cohorts of 121 BTKi-treated patients, 48 (39.7%) progressed on treatment with BTK and/or PLCG2 mutations detected in 87% of CLL progressions. Consistent with a recent report, homogeneous and bimodal CD49d-positive cases (the latter having concurrent CD49d+ and CD49d− CLL subpopulations, irrespective of the traditional 30% cutoff value) had a shorter time to progression of 6.6 years, whereas 90% of cases homogenously CD49d− were estimated progression-free at 8 years (P = 0.0004).Conclusions:CD49d/VLA-4 emerges as a microenvironmental factor that contributes to BTKi resistance in CLL. The prognostic value of CD49d is improved by considering bimodal CD49d expression.

Supplementary Figure 2 Legend from Circulating B-Cell Chronic Lymphocytic Leukemia Cells Display Impaired Migration to Lymph Nodes and Bone Marrow

Supplementary Table 2 from Circulating B-Cell Chronic Lymphocytic Leukemia Cells Display Impaired Migration to Lymph Nodes and Bone Marrow

Supplementary Figure 1 Legend from Circulating B-Cell Chronic Lymphocytic Leukemia Cells Display Impaired Migration to Lymph Nodes and Bone Marrow

Supplementary Table 2 Legend from Circulating B-Cell Chronic Lymphocytic Leukemia Cells Display Impaired Migration to Lymph Nodes and Bone Marrow

Supplementary Table 1 from Modifying Akt Signaling in B-Cell Chronic Lymphocytic Leukemia Cells

Supplementary Table 1 from Circulating B-Cell Chronic Lymphocytic Leukemia Cells Display Impaired Migration to Lymph Nodes and Bone Marrow

Supplementary Figures 1-5 from Modifying Akt Signaling in B-Cell Chronic Lymphocytic Leukemia Cells

Supplementary Table 1 Legend from Circulating B-Cell Chronic Lymphocytic Leukemia Cells Display Impaired Migration to Lymph Nodes and Bone Marrow

Supplementary Figure 1 from Circulating B-Cell Chronic Lymphocytic Leukemia Cells Display Impaired Migration to Lymph Nodes and Bone Marrow

Supplementary Methods from Modifying Akt Signaling in B-Cell Chronic Lymphocytic Leukemia Cells

PDF file - 245K, Supplementary Figure S1: Activated, autologous T cells induce CLL cell proliferation. Supplementary Figure S2: CD40L activated CLL cells fully retain their chemotactic response towards CCL21. Supplementary Figure S3: PMA/ionomycin and IgM cross-linking induces FL-HA binding. Supplementary Figure S4: CD44 surface expression on CLL patients does not differ between clinical risk groups. Supplementary Figure S5: The CLL cell subpopulation remaining CD44v6 negative after CD40L-activation displays low activation levels. Supplementary Figure S6: The mAbs used for immunoblotting specifically recognize panCD44 and CD44v6

Supplementary Figure 2 from Circulating B-Cell Chronic Lymphocytic Leukemia Cells Display Impaired Migration to Lymph Nodes and Bone Marrow

Emerging evidence suggests that the survival of B-cell chronic lymphocytic leukemia (CLL) cells is dependent on microenvironmental influences such as antigenic stimulation and support by stromal cells. Akt, also known as protein kinase B, is a central component in prosurvival signaling downstream of these events. We investigated the role of Akt and its modulation by the protooncogene T-cell leukemia 1a (Tcl1a) in the survival pathways of primary CLL samples and CLL-derived prolymphocytic cell lines MEC-1 and MEC-2. Akt activation was increased by the protective presence of human bone marrow stromal cells and B-cell receptor mimicking signals but antagonized by direct Akt blockade with the novel specific inhibitor AiX, with preferential apoptosis induction in CLL cells with an unmutated immunoglobulin status, which predicts poor clinical outcome. In addition, we found a direct interaction of Akt with Tcl1a in an endogenous coimmunoprecipitation assay. Confirming the critical role of Tcl1a in modulating Akt signaling, Akt activation was enhanced by overexpressing Tcl1a in CLL. In contrast, decreasing Tcl1a levels by small interfering RNA reduced Akt activation in the fludarabine-insensitive CLL cell line MEC-2 and sensitized the malignant cells to fludarabine treatment. In summary, our data reveal a significant role for the Akt-Tcl1a axis in CLL survival and propose a further evaluation of this interplay for targeting chemoresistance phenomena. Cancer Res; 70(18); 7336–44. ©2010 AACR.

Homing to secondary lymphoid organs and bone marrow (BM) is a central aspect of leukemic pathophysiology. We investigated the roles of the two major lymphocyte integrins LFA-1 and VLA-4 on B-cell chronic lymphocytic leukemia (CLL) cells in these processes. We found that the majority of CLL cells expressed significantly reduced LFA-1 due to low β2 integrin transcripts. VLA-4 expression was heterogenous but underwent rapid activation by the BM chemokine CXCL12. CLL cells failed to transmigrate across VCAM-1–expressing, ICAM-1–expressing, and CXCL12-expressing endothelium, whereas when LFA-1 expression was regained in subsets of CLL cells, these lymphocytes rapidly transmigrated the endothelium. Furthermore, when injected into tail veins of immunodeficient mice, normal B cells rapidly homed to lymph nodes (LN) in a LFA-1–dependent manner, whereas CLL cells did not. Nevertheless, only residual CLL subsets could reenter BM, whereas both normal and CLL cells homed to the mice spleen in an LFA-1–independent and VLA-4–independent manner. Our results suggest that CLL cells have a reduced capacity to adhere and transmigrate through multiple vascular endothelial beds and poorly home to lymphoid organs other than spleen. Integrin blocking could thus be an efficient strategy to prevent circulating CLL cells from reaching prosurvival niches in LNs and BM but not in spleen. [Cancer Res 2009;69(7):OF3121–30]

CD49d, the α4 chain of the VLA-4 integrin, is a negative prognosticator in chronic lymphocytic leukemia (CLL) with a key role in CLL cell-microenvironment interactions mainly occurring via its ligands VCAM-1 and fibronectin. In the present study, we focused on EMILIN-1 (Elastin-MIcrofibriL-INterfacer-1), an alternative VLA-4 ligand whose role has been so far reported only in non-hematological settings, by investigating: i) the distribution of EMILIN-1 in CLL-involved tissues; ii) the capability of EMILIN-1 to operate, via its globular C1q (gC1q) domain, as additional adhesion ligand in CLL; iii) the functional meaning of EMILIN-1 gC1q/VLA-4 interactions in CLL. EMILIN-1 is widely present in the CLL-involved areas of bone marrow biopsies (BMBs) without difference between CD49d negative and positive cases, displaying at least three different expression patterns: "fibrillar", "dot-like" and "mixed". The lack in CLL-BMB of neutrophil elastase, whose proteolytic activity degrades EMILIN-1 and impairs EMILIN-1 function, suggests full functional EMILIN-1 in CLL independently of its expression pattern. Functionally, EMILIN-1 gC1q domain promotes adhesion of CLL cells through specific interaction with VLA-4, and releases pro-survival signals for CLL cells, as demonstrated by enhanced ERK and AKT phosphorylation and impairment of in-vitro-induced apoptosis. EMILIN-1/VLA-4 interaction can efficiently contribute to the maintenance of the neoplastic clone in CLL. This article is protected by copyright. All rights reserved.

Acute myeloid leukemia (AML) has a poor prognosis under the current standard of care. In recent years, venetoclax, a BCL-2 inhibitor, was approved to treat patients who are ineligible for intensive induction chemotherapy. However, complete remission rates with venetoclax-based therapies are hampered by minimal residual disease (MRD) in a proportion of patients, leading to relapse. MRD is a result of leukemic stem cells being retained in bone marrow protective environments; activation of the CXCL12-CXCR4 pathway was shown to be relevant to this process. An important role is also played by cell adhesion molecules such as CD44, which has been shown to be crucial for the development of AML. Here we show that CD44 is involved in CXCL12 promotion of resistance to venetoclax-induced apoptosis in human AML cell lines and AML patient samples, which could be abrogated by CD44 knock down, knockout, or blocking with an anti-CD44 antibody. Split-Venus bimolecular fluorescence complementation showed that CD44 and CXCR4 physically associate at the cell membrane upon CXCL12 induction. In the venetoclax-resistant OCI-AML3 cell line, CXCL12 promoted an increase in the proportion of cells expressing high levels of embryonic stem cell core transcription factors (ESC-TFs: Sox2, Oct4, Nanog) abrogated by CD44 knockdown. This ESC-TF–expressing subpopulation which could be selected by venetoclax treatment, exhibited a basally enhanced resistance to apoptosis and expressed higher levels of CD44. Finally, we developed a novel AML xenograft model in zebrafish, which showed that CD44 knockout sensitizes OCI-AML3 cells to venetoclax treatment in vivo. Our study shows that CD44 is a potential molecular target for sensitizing AML cells to venetoclax-based therapies.

Integrin-mediated interactions between hematopoietic cells and their microenvironment are important for the development and function of immune cells. Here, the role of the integrin adaptor Kindlin-3 in B cell homeostasis is studied. Comparing the individual steps of B cell development in B cell-specific Kindlin-3 or alpha4 integrin knockout mice, we found in both conditions a phenotype of reduced late immature, mature, and recirculating B cells in the bone marrow. In the spleen, constitutive B cell-specific Kindlin-3 knockout caused a loss of marginal zone B cells and an unexpected expansion of follicular B cells. Alpha4 integrin deficiency did not induce this phenotype. In Kindlin-3 knockout B cells VLA-4 as well as LFA-1-mediated adhesion was abrogated, and short-term homing of these cells in vivo was redirected to the spleen. Upon inducible Kindlin-3 knockout, marginal zone B cells were lost due to defective retention within 2 weeks, while follicular B cell numbers were unaltered. Kindlin-3 deficient follicular B cells displayed higher IgD, CD40, CD44, CXCR5, and EBI2 levels, and elevated PI3K signaling upon CXCR5 stimulation. They also showed transcriptional signatures of spontaneous follicular B cell activation. This activation manifested in scattered germinal centers in situ, early plasmablasts differentiation, and signs of IgG class switch.

The bone marrow microenvironment provides critical cues for hematopoietic stem cell (HSC) self-renewal and differentiation and contributes to their malignant conversion. The microenvironment comprises a complex mixture of multiple cell types, soluble factors, and extracellular matrix in specialized regions termed ‘niches.’ Positioning of the various cellular players within these niches depends on their repertoire of adhesion molecules and chemotactic signaling, involving integrins and chemokine receptors and the corresponding intracellular players such as kinases and GTPases. The mechanical role of adhesion is to control the strength and morphology of the cell-cell and cell-extracellular matrix contacts and thereby the energy needed for the optimal localization of cells to their surroundings. While it is clear that biomechanical adhesive bonds are energetically expensive, the crosstalk between cell adhesion and metabolic pathways in the normal and malignant microenvironment is far from understood. The metabolic profile of the various cell types within the niche includes key molecules such as AMPK, glucose, mTOR, and HIF-1α. Here, we describe our most recent understanding of how the interplay between adhesion and these metabolic components is indispensable for bone marrow niche stability. In parallel, we compare the altered crosstalk of different cell types within the bone marrow niches in hematological malignancies and propose potential therapeutic associations.

Aberrant activation of the NLR family pyrin domain containing 3 (NLRP3) inflammasome mediates numerous inflammatory diseases. Oncogenes can activate the NLRP3 inflammasome and thereby promote myeloproliferative neoplasia, suggesting a crucial role of NLRP3 in the malignant transformation of hematopoietic cells. Here, we show that bone marrow-derived mononuclear cells of AML patients display enhanced expression of NLRP3, IL-1β and IL-18 and that high-level expression of NLRP3 is linked to poor survival of AML patients. Pharmacological and genetic inhibition of NLRP3 inflammasome activation attenuated cell proliferation of MOLM-13 AML cells in vitro. In vivo, genetic inhibition of NLRP3 in MOLM-13 AML cells resulted in reduced engraftment potential in xenografts, along with reduced splenomegaly and organ infiltration. Differential proteomic analysis revealed the eIF2 pathway as potential target of NLRP3 in AML, with a significant increase of eIF2α phosphorylation upon NLRP3 inhibition. NLRP3 inhibition also caused a strong decrease in cyclin – dependent kinases CDK4 and CDK6, accompanied by an upregulation of the CDK inhibitor p21 (CDKN1A) and a marked arrest of cell cycle progression in the G0/G1 phase, consistent with the role of eIF2α phosphorylation as negative cell cycle regulator.Taken together, we show that inhibition of the NLRP3 inflammasome reduces AML cell proliferation by promoting eIF2α phosphorylation, which in turn enhances the expression of cell cycle arrest genes such as p21. Thus, the study uncovers the NLRP3/eIF2 axis as new driver of AML proliferation and proposes a novel therapeutic treatment of AML by targeted inhibition of NLRP3 activation.

Lineage commitment and differentiation of hematopoietic cells takes place in well-defined microenvironmental surroundings. Communication with other cell types is a vital prerequisite for the normal functions of the immune system, while disturbances in this communication support the development and progression of neoplastic disease. Integrins such as the integrin very late antigen-4 (VLA-4; CD49d/CD29) control the localization of healthy as well as malignant B cells within the tissue, and thus determine the patterns of organ infiltration. Malignant B cells retain some key characteristics of their normal counterparts, with B cell receptor (BCR) signaling and integrin-mediated adhesion being essential mediators of tumor cell homing, survival and proliferation. It is thus not surprising that targeting the BCR pathway using small molecule inhibitors has proved highly effective in the treatment of B cell malignancies. Attenuation of BCR-dependent lymphoma–microenvironment interactions was, in this regard, described as a main mechanism critically contributing to the efficacy of these agents. Here, we review the contribution of VLA-4 to normal B cell differentiation on the one hand, and to the pathophysiology of B cell malignancies on the other hand. We describe its impact as a prognostic marker, its interplay with BCR signaling and its predictive role for novel BCR-targeting therapies, in chronic lymphocytic leukemia and beyond.

Adhesive properties of leukemia cells shape the degree of organ infiltration and the extent of leukocytosis. CD44 and the integrin VLA-4, a CD49d/CD29 heterodimer, are important factors of progenitor cell adhesion in bone marrow (BM). Here, we report their cooperation in acute myeloid leukemia (AML) by a novel non-classical CD44-mediated way of inside-out VLA-4 activation. In primary AML BM samples from patients and the OCI-AML3 cell line, CD44 engagement by hyaluronan induced inside-out activation of VLA-4 resulting in enhanced leukemia cell adhesion on VCAM-1. This was independent from VLA-4 affinity regulation but based on ligand-induced integrin clustering on the cell surface. CD44-induced VLA-4 activation could be inhibited by the Src family kinase inhibitor PP2 and the multikinase inhibitor midostaurin. In further consequence, the increased adhesion on VCAM-1 allowed AML cells to strongly bind stromal cells. Thereby VLA-4/VCAM-1 interaction promoted activation of Akt, MAPK, NF-kB and mTOR signaling and decreased AML cell apoptosis. Collectively, our investigations provide a mechanistic description of an unusual CD44 function in regulating VLA-4 avidity in AML, supporting AML cell retention in the supportive BM microenvironment.

Despite recent advances, chemoimmunotherapy remains a standard for fit previously untreated chronic lymphocytic leukaemia patients. Lenalidomide had activity in early monotherapy trials, but tumour lysis and flare proved major obstacles in its development. We combined lenalidomide in increasing doses with six cycles of fludarabine and rituximab (FR), followed by lenalidomide/rituximab maintenance. In 45 chemo-naive patients, included in this trial, individual tolerability of the combination was highly divergent and no systematic toxicity determining a maximum tolerated dose was found. Grade 3/4 neutropenia (71%) was high, but only 7% experienced grade 3 infections. No tumour lysis or flare > grade 2 was observed, but skin toxicity proved dose-limiting in nine patients (20%). Overall and complete response rates after induction were 89 and 44% by intention-to-treat, respectively. At a median follow-up of 78.7 months, median progression-free survival (PFS) was 60.3 months. Minimal residual disease and immunoglobulin variable region heavy chain mutation state predicted PFS and TP53 mutation most strongly predicted OS. Baseline clinical factors did not predict tolerance to the immunomodulatory drug lenalidomide, but pretreatment immunophenotypes of T cells showed exhausted memory CD4 cells to predict early dose-limiting non-haematologic events. Overall, combining lenalidomide with FR was feasible and effective, but individual changes in the immune system seemed associated with limiting side effects. clinicaltrials.gov (NCT00738829) and EU Clinical Trials Register (www.clinicaltrialsregister.eu, 2008-001430-27) Electronic supplementary material The online version of this article (10.1007/s00277-018-3380-z) contains supplementary material, which is available to authorized users.

Tissino et al. demonstrate that in chronic lymphocytic leukemia, the VLA-4 (CD49d/CD29) integrin remains activable by B cell receptor stimulation also upon in vitro and in vivo ibrutinib exposure. Clinically, ibrutinib-treated CD49d-positive CLL patients experience reduced recirculation lymphocytosis and nodal response and inferior outcomes., The Bruton’s tyrosine kinase (BTK) inhibitor ibrutinib, which antagonizes B cell receptor (BCR) signals, demonstrates remarkable clinical activity in chronic lymphocytic leukemia (CLL). The lymphocytosis experienced by most patients under ibrutinib has previously been attributed to inhibition of BTK-dependent integrin and chemokine cues operating to retain the tumor cells in nodal compartments. Here, we show that the VLA-4 integrin, as expressed by CD49d-positive CLL, can be inside-out activated upon BCR triggering, thus reinforcing the adhesive capacities of CLL cells. In vitro and in vivo ibrutinib treatment, although reducing the constitutive VLA-4 activation and cell adhesion, can be overcome by exogenous BCR triggering in a BTK-independent manner involving PI3K. Clinically, in three independent ibrutinib-treated CLL cohorts, CD49d expression identifies cases with reduced lymphocytosis and inferior nodal response and behaves as independent predictor of shorter progression-free survival, suggesting the retention of CD49d-expressing CLL cells in tissue sites via activated VLA-4. Evaluation of CD49d expression should be incorporated in the characterization of CLL undergoing therapy with BCR inhibitors.

Introduction The development and maturation of B cells is highly dependent on signals provided by the microenvironment of the lymphatic organs. As B cells move from one developmental stage and niche to the next, the integrin family of adhesion molecules provides important cues for their correct positioning and retention. The integrin adaptor protein Kindlin-3 (encoded by the Fermt3 gene) regulates integrin activity and function in a wide range of hematopoietic cell types. In this study, we aimed to define its precise role in the development and function of the different murine B cell subsets. Methods We crossed a Fermt3flox/flox mb1-cre mouse strain (hereforth called K3ΔB mice), harboring a B cell specific Kindlin-3 deletion. B cell subsets in the different lymphoid organs of these K3ΔB mice and control littermates were defined by multicolor flow cytometry. Adoptive transfer, microscopy and real-time flow cytometry were used to analyze the different steps of integrin activation. A co-culture system with OP9 stromal cells and BAFF was used to assess the in vitro differentiation potential of immature progenitors into the different mature B cell subsets. Transcriptional differences between follicular B cells isolated from spleens of K3ΔB- and control mice were assessed by transcriptome array. Results In vitro, we found that integrin activation on B cells was induced upon activation of the chemokine receptors CXCR4 and CXCR5 or the B cell receptor. This stimulation triggered adhesion of wild type B cells to integrin ligands under shear flow. The increase of VLA-4 integrin affinity to its ligand substrates during this process could also be calculated from real-time flow cytometrical analyses. In contrast, K3ΔB-derived B cells could not reach high affinity states of integrins and thus failed to adhere on the substrates upon stimulation, despite slight upregulation of chemokine receptors CXCR4 and CXCR5. B cell migration towards the respective chemokines also required Kindlin-3, even in an integrin ligand-free setting. In vivo, Kindlin-3 was required for homing of mature B cells to the bone marrow and to lymph nodes. When further characterizing K3ΔB mice by flow cytometry and immunohistochemistry we observed increased early B cell numbers in the bone marrow. Of note, marginal zone (MZ) B cells in the spleen were completely absent (Figure 1 A+B). We consequently assessed the potential of immature B cells to develop into B cells with high expression of CD21, a marker for MZ B cells, upon their co-culture with OP9 stromal cells in the presence of the B cell survival factor BAFF. While 18% of B cells differentiating from wild type bone marrow displayed high expression of CD21, the percentage of CD21 high cells recovered from Kindlin-3 deficient progenitors was significantly lower (~12%, Figure 1C). Pathways involved in these developmental differences were analyzed by a transcriptome array, revealing increased activity of the B cell receptor pathway in the knockout situation accompanied by higher, NFkappaB and Notch signaling. Conclusion/Outlook Whereas our results highlight the importance of Kindlin-3 dependent, integrin mediated cell retention and migration during B cell development they also indicate that Kindlin-3 functions in an integrin-independent manner when regulating cell motility and transcription. The complete lack of MZ B cells in the absence of Kindlin-3 is thus most likely a combination of defective retention in the MZ area and transcriptional alterations favoring the development of transitional B cells into follicular- rather than MZ B cells. Figure 1 : B-cell specific Kindlin-3 knockout leads to loss of splenic marginal zone B cells. The percentage of MZ B-cells among total splenic B cells was determined by flow cytometry in K3ΔB mice and wild type (wt) littermates (A). Immunohistochemistry staining of CD19 showed a loss of loosely packed marginal zone B cells (yellow arrows) in the absence of Kindlin-3 (B). B cells were enriched from the bone marrow of K3ΔB mice and wt littermates and cultured on a confluent layer of OP9 cells in the presence of 200 ng/ml BAFF for 72 h. Development of CD21 high/CD23 low B cells was then determined by flow cytometry (C). Figure Disclosures Greil: Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, accomodations, expenses, Research Funding; Astra zeneca: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, accomodations, expenses, Research Funding; Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, accomodations, expenses, Research Funding; Daiichi Sankyo, Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, accomodations, expenses, Research Funding; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, accomodations, expenses, Research Funding; F. Hoffmann-La Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, accomodations, expenses, Research Funding; BMS/celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, accomodations, expenses, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, accomodations, expenses, Research Funding; MSD Merck: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, accomodations, expenses, Research Funding; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, accomodations, expenses, Research Funding.

The integrin heterodimer CD49d/CD29 (a.k.a. Very Late Antigen-4, VLA-4) mediates cell-cell and cell-matrix interaction through the binding of its ligands VCAM-1 and fibronectin. VLA-4 can be present on the cell surface at different conformation states that affect the binding affinity for the ligands. In chronic lymphocytic leukemia (CLL), higher VLA-4 levels, as determined by measuring the expression of CD49d chain by flow cytometry, have been demonstrated to associate with a worse prognosis, in keeping with the role of VLA-4 as key molecule favoring CLL cell localization in protective niches of bone marrow and lymph nodes. Given the emerging clinical relevance of VLA-4 evaluation in CLL, both in the setting of the conventional chemo-immunotherapy and the novel drugs targeting the BCR pathway, here we describe the flow cytometric approaches followed by us to quantify the CD49d expression levels and the VLA-4 activation status in CLL cells.

The integrin heterodimer CD49d/CD29 (a.k.a. Very Late Antigen-4, VLA-4) mediates cell-cell and cell-matrix interaction through the binding of its ligands VCAM-1 and fibronectin. VLA-4 can be present on the cell surface at different conformation states that affect the binding affinity for the ligands. In chronic lymphocytic leukemia (CLL), higher VLA-4 levels, as determined by measuring the expression of CD49d chain by flow cytometry, have been demonstrated to associate with a worse prognosis, in keeping with the role of VLA-4 as key molecule favoring CLL cell localization in protective niches of bone marrow and lymph nodes. Given the emerging clinical relevance of VLA-4 evaluation in CLL, both in the setting of the conventional chemo-immunotherapy and the novel drugs targeting the BCR pathway, here we describe the flow cytometric approaches followed by us to quantify the CD49d expression levels and the VLA-4 activation status in CLL cells.

// Thomas Melchardt 1, 2, 3, * , Clemens Hufnagl 1, 2, 3, * , David M. Weinstock 4 , Nadja Kopp 4 , Daniel Neureiter 5 , Wolfgang Trankenschuh 5 , Hubert Hackl 6 , Lukas Weiss 1, 2, 3 , Gabriel Rinnerthaler 1, 2, 3 , Tanja N. Hartmann 1, 2, 3 , Richard Greil 1, 2, 3 Oliver Weigert 7, 8 , Alexander Egle 1, 2, 3 1 Third Medical Department at The Paracelsus Medical University Salzburg, Salzburg, Austria 2 Salzburg Cancer Research Institute (SCRI), Salzburg, Austria 3 Cancer Cluster Salzburg (CCS), Salzburg, Austria 4 Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA 5 Institute of Pathology, Paracelsus Medical University Salzburg, Salzburg, Austria 6 Division of Bioinformatics, Biocenter, Medical University of Innsbruck, Innsbruck, Austria 7 University Hospital of The Ludwig-Maximilians-University Munich, Medical Department III, Laboratory for Experimental Leukemia and Lymphoma Research (ELLF), Munich, Germany 8 German Cancer Consortium (DKTK) and German Cancer Research Center (DKFZ), Heidelberg, Germany * These authors have contributed equally to this work Correspondence to: Alexander Egle, email: a.egle@salk.at Keywords: DLBCL, clonal evolution, TP53, tumor heterogeneity, subclonal selection Received: November 18, 2015 Accepted: May 22, 2016 Published: June 06, 2016 ABSTRACT Little information is available about the role of certain mutations for clonal evolution and the clinical outcome during relapse in diffuse large B-cell lymphoma (DLBCL). Therefore, we analyzed formalin-fixed-paraffin-embedded tumor samples from first diagnosis, relapsed or refractory disease from 28 patients using next-generation sequencing of the exons of 104 coding genes. Non-synonymous mutations were present in 74 of the 104 genes tested. Primary tumor samples showed a median of 8 non-synonymous mutations (range: 0-24) with the used gene set. Lower numbers of non-synonymous mutations in the primary tumor were associated with a better median OS compared with higher numbers (28 versus 15 months, p=0.031). We observed three patterns of clonal evolution during relapse of disease: large global change, subclonal selection and no or minimal change possibly suggesting preprogrammed resistance. We conclude that targeted re-sequencing is a feasible and informative approach to characterize the molecular pattern of relapse and it creates novel insights into the role of dynamics of individual genes.

Chronic lymphocytic leukemia (CLL) is a heterogeneous disease with a variable clinical course. Novel biomarkers discovered over the past 20 years have revolutionized the way clinicians approach prognostication and treatment especially in the chemotherapy-free era. Herein, we review the best established prognostic and predictive biomarkers in the setting of chemoimmunotherapy (CIT) and novel targeted therapy. We propose that TP53 disruption (defined as either TP53 mutation or chromosome 17p deletion), unmutated immunoglobulin heavy chain variable region gene status (UM IGHV), NOTCH1 mutation, and CD49d expression are the strongest prognosticators of disease progression and overall survival in the field of novel biomarkers including recurrent gene mutations. We also highlight the predictive role of TP53 disruption, UM IGHV, and NOTCH1 mutation in the setting of CIT and TP53 disruption and CD49d expression in the setting of novel targeted therapy employing B-cell receptor (BCR) and B-cell lymphoma-2 (BCL2) inhibition. Finally, we discuss future directions in the field of biomarker development to identify those with relapsed/refractory disease at risk for progression despite treatment with novel therapies.