Study of bone metabolism in hematopoietic sten cell mobilization

Bone as a regulator of human hematopoietic stem cell (HSC) trafficking: Study of biochemical markers of bone remodeling and angiogenic cytokines during HSC mobilization, in patients candicates to autologous hematopoietic stem cell transplantation (AHSCT).Background: The mobilization and collection of peripheral blood stem cells (PBSC) is a standard procedure in the treatment of many hematologic malignancies, both in the autologous and allogeneic stem cell transplantation setting. The proposed mechanisms of HSC mobilization, based on animal models, implicate the G-CSF-mediated proteolytic cleavage of important ligands, such as stromal-derived factor-1 (SDF-1) and the suppression of these ligands on marrow stroma, and especially on osteoblasts (OB). These events abolish the HSC-niche adhesion and lead to HSC mobilization. Today, bone cells are not considered just as a structural, non-functional, supportive tissue for bone marrow, but as HSC-niche regulators. However, there are scarce data regarding the role of bone turnover in HSC mobilization in humans.Aim: To study biochemical markers of bone remodeling and angiogenic cytokines in the process of HSC mobilization of patients with lymphoma and multiple myeloma.Methods: Forty-six patients (18 with Non-Hodgkin lymphoma and 14 with multiple myeloma and Hodgkin lymphoma, respectively) were studied. Serum samples from each patient were collected at two different time points: before the initiation of mobilization (pre-mobilization sample) and on the day of PBSCcollection, which coincided with the highest circulating CD34 counts (collection sample). Moreover, 3 additional serum samples from 19/46 patients were collected, between mobilization and collection. The following molecules were measured by ELISA in patients’ sera: 1) bone resorption markers, carboxyterminal telopeptide of collagen type 1 (CTX), aminoterminal telopeptide of collagen type 1 (NTX) and tartrate resistant acid phosphatase isoform 5b (TRACP-5b), 2) bone formation markers, bone alkaline phosphatase (BALP), osteocalcin (OSC) and osteopontin (OPN), 3) the marker of osteocyte activity dickkopf-1 (DKK-1), an important inhibitor of Wnt-signalling and osteoblastogenesis 4) the osteoclastic regulators receptor activator NF-kB ligand (RANKL) and osteoprotegerin (OPG), 5) angiogenic cytokines angiopoietin-1 (ANGP1), angiopoietin-2 (ANGP2) and angiogenin (ANG), 6) cytokine IL-11, adhesion molecule intercellular adhesion molecule-1 (ICAM-1), chemokine SDF-1 and proteinoglycan Syndecan-1.Values between the two different time points were compared with nonparametric methods. Patients who had either a PBSC (CD34+cells) collection <2.0x106/kg, or a circulating CD34 count peak <20/μL were considered as “poor mobilizers”.Results: The comparison of the molecules under study between the premobilization and collection samples revealed the following: BALP (p=0.000) and OPN (p=0.049) increased significantly between pre-mobilization and collection. OSC, a marker of bone turnover, as well as DKK-1 decreased significantly (p=0.000 and p=0.041, respectively). These findings, all together reveal significant increase of bone formation during HSC mobilization. At the same time, there was a significant increase of RANKL (p=0.000) and OPG (p=0.000) levels, leading to an increase of RANKL/OPG ratio (p=0.000), showing osteoclastogenesis enchacement and osteoclastic activation. This finding was however most prominent in patients with myeloma. Interestingly, there was no evidence of increased osteoclastic activity, as CTX decreased significantly (p=0.026), while both NTX and TRACP-5b did not change. ANGP-1 showed a dramatic reduction (p=0.000), while ANGP-2 increased (p=0.000), resulting in a significant decrease of the ANGP-1/ANGP-2 ratio, a finding indicating significant vessel destabilization during mobilization. The aforementioned results were further supported by the intermediate measurements, which showed a straightforward alteration of bone metabolism early in HSC mobilization. The comparison between good and poor mobilizers revealed that poor mobilizers had significantly higher CTX levels both at pre-mobilization (p=0.004) and collection samples (p=0.001), higher NTX levels at collection (p=0.02), lower ANGP-1 pre-mobilization (p=0.004) and higher OSC at collection (p=0.000) compared to good mobilizers. It seems that CTX, NTX and ANGP-1 premobilization levels may serve as reliable predictors of poor mobilization.Summary/Conclusions: Our study showed for the first time that bone plays adynamic role during human HSC mobilization: Bone formation and vessel destabilization are the two major events and osteoblasts seem to be the major orchestrating cells during this process. Osteoclasts are stimulated, but not fully active. Moreover, several of these markers may identify poor mobilizers.Ο οστίτης ιστός ως ρυθμιστής της ανθρώπινης κινητοποίησης των αρχέγονων αιμοποιητικών αιμοποιητικών κυττάρων (hematopoietic stem cells-HSC): Δείκτες οστικού μεταβολισμού και αγγειογενετικές κυττοκίνες στην HSC κινητοποίηση ασθενών, υποβαλλόμενων σε αυτόλογη μεταμόσχευση αρχέγονων αιμοποιητικών κυττάρων (ΑΜΑΑΚ).Εισαγωγή: Η κινητοποίηση και συλλογή των HSC του αίματος θεωρείται βασική θεραπευτική στρατηγική σε αρκετές αιματολογικές κακοήθειες που χρήζουν ΑΜΑΑΚ. Οι προτεινόμενοι μηχανισμοί κινητοποίησης των HSC, από τη νησίδα του μυελού των οστών που κατοικούν στην κυκλοφορία, στηρίζονται σε μελέτες σε πειραματόζωα και εμπλέκουν την G-CSF-επαγόμενη πρωτεόλυση κρίσιμων συνδετών, όπως ο παράγοντας-1 προερχόμενος από κύτταρα του στρώματος (SDF-1), και τη μείωση έκφρασης των συνδετών αυτών από το μυελικό στρώμα και ειδικά τους οστεοβλάστες (ΟΒ). Τα γεγονότα αυτά αίρουν την προσκόλληση των HSC στη νησίδα και τα οδηγούν σε κινητοποίηση. Πλέον ο οστίτης ιστός δεν θεωρείται απλά ένας δομικός, μη λειτουργικός, στηρικτικός ιστός του μυελού των οστών αλλά ρυθμιστής της HSC νησίδας, πλην όμως, υπάρχουν ελάχιστες πληροφορίες αναφορικά με το ρόλο της οστικής ανακατασκευής στην ανθρώπινη HSC κινητοποίηση.Σκοπός: Η μελέτη δεικτών του οστικού μεταβολισμού και αγγειογενετικών κυττοκινών στην HSC κινητοποίηση ασθενών με λέμφωμα ή πολλαπλό μυέλωμα.Μέθοδοι: Μελετήθηκαν 46 ασθενείς (18 με Non-Hodgkin λέμφωμα και 14 με πολλαπλό μυέλωμα και Hodgkin λέμφωμα αντίστοιχα). Συλλέχθηκε ορός από κάθε ασθενή σε δύο κομβικές χρονικές στιγμές: προ της έναρξης της κινητοποίησης και την ημέρα της συλλογής των HSC. Επιπλέον συλλέχθηκαν και 3 ενδιάμεσα, αλληλοδιάδοχα δείγματα ορού κατά τη διάρκεια της κινητοποίησης. Μετρήθηκαν με ELISA τα ακόλουθα μόρια στον ορό τωνασθενών: 1) Δείκτες οστικής απορρόφησης: CTX και NTX (τελοπεπτίδια-προιόντα απορρόφησης κολλαγόνου τύπου Ι μέσω καθεψίνης Κ των οστεοκλαστών) και TRACP-5b (ισομορφή 5b της όξινης φωσφατάσης ανθεκτικής στο τρυγικό οξύ, ένζυμο των οστεοκλαστών που αποδομεί την εξωκυττάρια θεμέλια ουσία), 2) Δείκτες οστικής δόμησης / ανακατασκευής: οστική αλκαλική φωσφατάση (BALP), οστεοκαλσίνη (OSC) και οστεοποντίνη (OPN), 3) Δείκτης δραστηριότητας οστεοκυττάρων και αναστολέας οστεοβλαστογένεσης / οστικής δόμησης: dickkopf-1 (DKK-1), 4) Δείκτες δραστηριότητας οστεοκυττάρων και OB και ρυθμιστές οστεοκλαστών: συνδέτης του υποδοχέα-ενεργοποιητή πυρηνικού παράγοντα κ-ελαφράς αλυσίδας, ευοδωτή ενεργοποιημένων Β-κυττάρων (RANKL) και οστεοπροτεγερίνη (OPG), 5) Δείκτες αγγειογένεσης: αγγειογενίνη (ANG), αγγειοποιητίνη-1 και -2 (ANGP-1 και ANGP-2), 6) Η κυττοκίνη ιντερλευκίνη-11 (IL-11), το διακυτταρικό κυτταρικό μόριο προσκόλλησης 1 (ICAM-1), η χημοκίνη SDF-1 και η πρωτεϊνογλυκάνη syndecan-1 (SYND-1). Οι τιμές των δειγμάτων συγκρίθηκαν με μη παραμετρικές μεθόδους. Ασθενείς στους οποίους συλλέχθηκαν CD34+ κύτταρα <2,0x106/kg ή/και η μέγιστη τιμή των κυκλοφορούντων CD34+ κυττάρων ήταν <20/μL θεωρήθηκαν «πτωχοί κινητοποιητές».Αποτελέσματα: Η σύγκριση των μορίων μεταξύ των δύο χρονικών στιγμών (δείγμα προ κινητοποίησης και δείγμα συλλογής) ανέδειξε τα παρακάτω: Η BALP (p=0.000) και η OPN (p=0.049) αυξάνονται στατιστικά σημαντικά μεταξύ προ κινητοποίησης και συλλογής. Η OSC, δείκτης οστικής ανακατασκευής, μειώνεται (p=0.000), όπως και ο DKK-1 (p=0.041), ο οποίος ως κρίσιμος μεταγραφικός συν-παράγοντας για την έκφραση της OSC στους ώριμους ΟΒ,ίσως εξηγεί την πτώση της. Τα δεδομένα αυτά καταδεικνύουν σημαντική αύξηση της οστικής δόμησης κατά την κινητοποίηση. Ο RANKL (p=0.000) και η OPG (p=0.000) αυξάνονται σημαντικά, οδηγώντας περαιτέρω σε σημαντική αύξηση του λόγου RANKL/OPG (p=0.000), αναδεικνύοντας ευόδωση οστεοκλαστογένεσης και ενεργοποίησης οστεοκλαστών κατά την κινητοποίηση, εύρημα κυρίως στους ασθενείς με πολλαπλό μυέλωμα. Πλην όμως αύξησηοστεοκλαστικής δραστηριότητας και οστικής απορρόφησης δεν απεδείχθη εφόσον το CTX μειώθηκε (p=0.026) ενώ το NTX και η TRACP-5b δεν μεταβλήθηκαν. Η ANGP-1 μειώθηκε δραματικά (p=0.000) ενώ η ANGP-2 αυξήθηκε (p=0.000), οδηγώντας σε σημαντική πτώση του λόγου ANGP-1/ANGP-2 (p=0.000), γεγονός που υποδηλώνει έντονη αγγειακή αποσταθεροποίηση κατά την HSC κινητοποίηση. Οι μετρήσεις των ενδιάμεσων ευρημάτων ανέδειξαν ενεργοποίηση του οστικού μεταβολισμού 2-4 ημέρες από την έναρξη της κινητοποίησης και κατοχύρωσαν τα προαναφερόμενα αποτελέσματα. Η σύγκριση μεταξύ καλών και πτωχών κινητοποιητών κατέδειξε, μεταξύ των άλλων, ότι οι πτωχοί κινητοποιητές είχαν στατιστικώς σημαντικά υψηλότερα επίπεδα CTX τόσο στο δείγμα προ κινητοποίησης (p=0.004) όσο και στο δείγμα συλλογής (p=0.001), υψηλότερα επίπεδα NTX στη συλλογή (p=0.02), χαμηλότερα επίπεδα ANGP-1 προ κινητοποίησης (p=0.004) και υψηλότερα επίπεδα OC στη συλλογή (p=0.000) σε σύγκριση με τους καλούς κινητοποιητές. Τα επίπεδα CTX, NTX και ANGP-1 προ κινητοποίησης φαίνεται να διακρίνουν στατιστικά την πτωχή κινητοποίηση.Συμπεράσματα: Τα αποτελέσματα της μελέτης μας δείχνουν για πρώτη φορά ότι η αύξηση της οστεοβλαστικής δραστηριότητας/οστικής δόμησης και η αγγειακή αποσταθεροποίηση αποτελούν σημαντικά γεγονότα στην ανθρώπινη HSC κινητοποίηση, καταδεικνύοντας το δυναμικό ρόλο του οστίτη ιστού. Ενώ η ενεργοποίηση των οστεοκλαστών συμβαίνει στην HSC κινητοποίηση, δεν συνοδεύεται από αύξηση οστικής απορρόφησης. Επιπλέον, κάποιοι από τους δείκτες οστικού μεταβολισμού και αγγειογένεσης μπορούν να χρησιμοποιηθούν στην αναγνώριση των πτωχών κινητοποιητών

New Insights in the Mobilization of Hematopoietic Stem Cells in Lymphoma and Multiple Myeloma Patients

Following chemotherapy and/or the administration of growth factors, such as granulocyte-colony stimulated factor (G-CSF), hematopoietic stem cells (HSC) mobilize from bone marrow to peripheral blood. This review aims to systematically present the structure of the HSC “niche” and elucidate the mechanisms of their mobilization. However, this field is constantly evolving and new pathways and molecules have been shown to contribute to the mobilization process. Understanding the importance and the possible primary pathophysiologic role of each pathway is rather difficult, since they share various overlapping components. The primary initiating event for the mobilization of HSC is chemotherapy-induced endogenous G-CSF production or exogenous G-CSF administration. G-CSF induces proliferation and expansion of the myelomonocytic series, which leads to proteolytic enzyme activation. These enzymes result in disruption of various receptor-ligand bonds, which leads to the disanchorage of HSC from the bone marrow stroma. In everyday clinical practice, CXC chemokine receptor-4 (CXCR4) antagonists are now being used as mobilization agents in order to improve HSC collection. Furthermore, based on the proposed mechanisms of HSC mobilization, novel mobilizing agents have been developed and are currently evaluated in preclinical and clinical studies

Small Lymphocytic Lymphoma: Analysis of Two Cohorts Including Patients in Clinical Trials of the German Chronic Lymphocytic Leukemia Study Group (GCLLSG) or in Real-Life Outside of Clinical Trials

Background: Only few studies have focused exclusively on patients with small lymphocytic lymphoma (SLL). Patients and Methods: In the present report, 103 SLL patients were analyzed from both, clinical trials of the German Chronic Lymphocytic Leukemia Study Group and Greek centers, and emphasis was placed on the therapeutic strategy. The impact of lymph node characteristics, such as the presence of proliferation centers (PCs) on response and survival was also assessed. Results: SLL patients included in clinical trials were treated mostly with fludarabine-based regimens while those in reallife were staged and treated mostly as patients with low-grade lymphomas. Our analysis showed a trend for better survival for patients with SLL without detectable PCs. Conclusion: Patients with SLL outside of clinical trials are usually treated as cases of lymphoma. In addition, this analysis supports published data regarding the adverse prognostic value of the presence of PCs in lymphoid nodes in SLL

Immunohistochemical Analysis of IL-6, IL-8/CXCR2 Axis, Tyrp-STAT-3, and SOCS-3 in Lymph Nodes from Patients with Chronic Lymphocytic Leukemia: Correlation between Microvascular Characteristics and Prognostic Significance

A number of studies have looked into the pathophysiological role of angiogenesis in CLL, but the results have often been inconsistent. We aimed to gain direct insight into the angiogenic process in lymph nodes involved by CLL, focusing on proangiogenic cytokines and microvessel morphometry. The tissue levels of VEGF, Th-2 cytokines IL-6 and IL-8, IL-8 receptor CXCR2, and tyrosine p-STAT-3/SOCS-3 axis modulating cytokine expression were evaluated immunohistochemically in 62 CLL/SLL cases. Microvascular characteristics were evaluated by image analysis. Results were analyzed with regard to clinicopathological characteristics. Proliferation centers (PCs) were less well vascularised compared to non-PC areas. IL-8 and CXCR2 expression was distinctly uncommon as opposed to IL-6, VEGF and SOCS-3, which were detected in the vast majority of cases. The latter two molecule expressions were more pronounced in the PCs in ∼40% of the cases. p-STAT-3 immunoreactivity was recorded in 66.67% of the cases with a predilection for PCs. Microvessel morphometry was unrelated to proangiogenic cytokines, p-STAT-3, SOCS-3, or survival. Microvascular caliber and VEGF expression were higher in Binet stage A, whereasIL-6 expression was higher in stage C. VEGF and p-STAT-3 exerted a favorable effect on progression, which remained significant in multivariate analysis, thereby constituting potential outcome predictors in CLL patients

Prognostic implication of the absolute lymphocyte to absolute monocyte count ratio in patients with classical Hodgkin lymphoma treated with doxorubicin, bleomycin, vinblastine, and dacarbazine or equivalent regimens.

Low absolute lymphocyte count (ALC) to absolute monocyte count (AMC) ratio (ALC/AMC) is an independent prognostic factor in Hodgkin lymphoma (HL), but different cutoffs (1.1, 1.5, and 2.9) have been applied. We aimed to validate the prognostic significance of ALC/AMC in 537 homogenously treated (doxorubicin, bleomycin, vinblastine, and dacarbazine or equivalents ± radiotherapy) classical HL patients at various cutoffs. The median ALC/AMC was 2.24 (0.44-20.50). The median AMC was 0.653 × 10(9)/L (0.050-2.070). Lower ALC/AMC was associated with established markers of adverse prognosis. In total, 477 (89%), 418 (78%), and 189 (35%) patients had an ALC/AMC ratio of ≥1.1, ≥1.5, and ≥2.9; respectively; 20% had monocytosis (≥0.9 × 10(9)/L). Ten-year time to progression (TTP) was 77% versus 55% for patients with ALC/AMC ≥1.1 and <1.1 (p = .0002), 76% versus 68% for ALC/AMC ≥1.5 and <1.5 (p = .049), 77% versus 73% for ALC/AMC ≥2.9 and <2.9 (p = .35), and 79% versus 70% for ALC/AMC ≥2.24 and <2.24 (p = .08), respectively. In stages ΙΑ/ΙΙΑ and in patients ≥60 years old, ALC/AMC had no significant effect on TTP. In advanced stages, ALC/AMC was significant only at the cutoff of 1.1 (10-year TTP 67% vs. 48%; p = .016). In younger, advanced-stage patients, the differences were more pronounced. In multivariate analysis of TTP, ALC/AMC < 1.1 (p = .007) and stage IV (p < .001) were independent prognostic factors; ALC/AMC was independent of International Prognostic Score in another model. ALC/AMC was more predictive of overall survival than TTP. At the cutoff of 1.1, ALC/AMC had independent prognostic value in multivariate analysis. However, the prognostically inferior group comprised only 11% of patients. Further research is needed prior to the widespread use of this promising marker

Identification of Very Low-Risk Subgroups of Patients with Primary Mediastinal Large B-Cell Lymphoma Treated with R-CHOP

Background R-CHOP can cure approximately 75% of patients with primary mediastinal large B-cell lymphoma (PMLBCL), but prognostic factors have not been sufficiently evaluated yet. R-da- EPOCH is potentially more effective but also more toxic than R-CHOP. Reliable prognostic classification is needed to guide treatment decisions. Materials and Methods We analyzed the impact of clinical prognostic factors on the outcome of 332 PMLBCL patients &lt;= 65 years treated with R-CHOP +/- radiotherapy in a multicenter setting in Greece and Cyprus. Results With a median follow-up of 69 months, 5-year freedom from progression (FFP) was 78% and 5-year lymphoma specific survival (LSS) was 89%. On multivariate analysis, extranodal involvement (E/IV) and lactate dehydrogenase (LDH) &gt;= 2 times upper limit of normal (model A) were significantly associated with FFP; E/IV and bulky disease (model B) were associated with LSS. Both models performed better than the International Prognostic Index (IPI) and the age-adjusted IPI by Harrel’s C rank parameter and Akaike information criterion. Both models A and B defined high-risk subgroups (13%-27% of patients [pts]) with approximately 19%-23% lymphoma-related mortality. They also defined subgroups composing approximately one-fourth or one-half of the patients, with 11% risk of failure and only 1% or 4% 5-year lymphoma-related mortality. Conclusion The combination of E/IV with either bulky disease or LDH &gt;= 2 times upper limit of normal defined high-risk but not very-high-risk subgroups. More importantly, their absence defined subgroups comprising approximately one-fourth or one-half of the pts, with 11% risk of failure and minimal lymphoma-related mortality, who may not need more intensive treatment such as R-da-EPOCH. Implications for Practice By analyzing the impact of baseline clinical characteristics on outcomes of a large cohort of patients with primary mediastinal large B-cell lymphoma homogeneously treated with R-CHOP with or without radiotherapy, we developed novel prognostic indices which can aid in deciding which patients can be adequately treated with R-CHOP and do not need more intensive regimens such as R-da-EPOCH. The new indices consist of objectively determined characteristics (extranodal disease or stage IV, bulky disease, and markedly elevated serum lactate dehydrogenase), which are readily available from standard initial staging procedures and offer better discrimination compared with established risk scores (International Prognostic Index [IPI] and age-adjusted IPI)