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Intercellular communication via gap junctions has a fundamental role in regulating cell growth and tissue homeostasis, and its dysregulation may be involved in cancer development and radio- and chemotherapy resistance. Connexin43 (Cx43) is the most ubiquitously expressed gap junction channel protein in human tissues. Emerging evidence indicates that dysregulation of the sorting of Cx43 to lysosomes is important in mediating the loss of Cx43-based gap junctions in cancer cells. However, the molecular basis underlying this process is currently poorly understood. Here, we identified the E3 ubiquitin ligase ITCH as a novel regulator of intercellular communication via gap junctions. We demonstrate that ITCH promotes loss of gap junctions in cervical cancer cells, which is associated with increased degradation of Cx43 in lysosomes. The data further indicate that ITCH interacts with and regulates Cx43 ubiquitination and that the ITCH-induced loss of Cx43-based gap junctions requires its catalytic HECT (homologous to E6-AP C-terminus) domain. The data also suggest that the ability of ITCH to efficiently promote loss of Cx43-based gap junctions and degradation of Cx43 depends on a functional PY (PPXY) motif in the C-terminal tail of Cx43. Together, these data provide new insights into the molecular basis underlying the degradation of Cx43 and have implications for the understanding of how intercellular communication via gap junctions is lost during cancer development., (© 2024. The Author(s).)

Background: Immune checkpoint blockade with monoclonal antibodies targeting programmed death 1 (PD-1) and its ligand PD-L1 has played a major role in the rise of cancer immune therapy. We have identified naturally occurring self-reactive T cells specific to PD-L1 in both healthy donors and cancer patients. Stimulation with a PD-L1 peptide (IO103), activates these cells to exhibit inflammatory and anti-regulatory functions that include cytotoxicity against PD-L1-expressing target cells. This prompted the initiation of the present first-in-human study of vaccination with IO103, registered at clinicaltrials.org (NCT03042793)., Methods: Ten patients with multiple myeloma who were up to 6 months after high dose chemotherapy with autologous stem cell support, were enrolled. Subcutaneous vaccinations with IO103 with the adjuvant Montanide ISA 51 was given up to fifteen times during 1 year. Safety was assessed by the common toxicity criteria for adverse events (CTCAE). Immunogenicity of the vaccine was evaluated using IFNγ enzyme linked immunospot and intracellular cytokine staining on blood and skin infiltrating lymphocytes from sites of delayed-type hypersensitivity. The clinical course was described., Results: All adverse reactions to the PD-L1 vaccine were below CTCAE grade 3, and most were grade 1-2 injection site reactions. The total rate of adverse events was as expected for the population. All patients exhibited peptide specific immune responses in peripheral blood mononuclear cells and in skin-infiltrating lymphocytes after a delayed-type hypersensitivity test. The clinical course was as expected for the population. Three of 10 patients had improvements of responses which coincided with the vaccinations., Conclusion: Vaccination against PD-L1 was associated with low toxicity and high immunogenicity. This study has prompted the initiation of later phase trials to assess the vaccines efficacy., Clinical Trial Registration: clinicaltrials.org, identifier NCT03042793., (Copyright © 2020 Jørgensen, Klausen, Grauslund, Helleberg, Aagaard, Do, Ahmad, Olsen, Klausen, Breinholt, Hansen, Martinenaite, Met, Svane, Knudsen and Andersen.)

Gap junctions consist of arrays of intercellular channels that enable adjacent cells to communicate both electrically and metabolically. Gap junctions have a wide diversity of physiological functions, playing critical roles in both excitable and non-excitable tissues. Gap junction channels are formed by integral membrane proteins called connexins. Inherited or acquired alterations in connexins are associated with numerous diseases, including heart failure, neuropathologies, deafness, skin disorders, cataracts and cancer. Gap junctions are highly dynamic structures and by modulating the turnover rate of connexins, cells can rapidly alter the number of gap junction channels at the plasma membrane in response to extracellular or intracellular cues. Increasing evidence suggests that ubiquitination has important roles in the regulation of endoplasmic reticulum-associated degradation of connexins as well as in the modulation of gap junction endocytosis and post-endocytic sorting of connexins to lysosomes. In recent years, researchers have also started to provide insights into the physiological roles of connexin ubiquitination in specific tissue types. This review provides an overview of the advances made in understanding the roles of connexin ubiquitination in the regulation of gap junction intercellular communication and discusses the emerging physiological and pathophysiological implications of these processes.

Cancer immune therapy is now used routinely for the treatment of several solid malignancies, albeit just recently having entered the clinic for treatment of haematological malignancies. Several studies demonstrate that cancer immune therapy is a promising treatment modality for the latter. Especially treatment with chimeric antigen receptor T cells for acute lymphoblastic leukaemia and lymphoma is promising. Other promising treatment modalities are immune check point inhibitors for both lymphoid and myeloid malignancies, as well as therapeutic cancer vaccination targeting tumour antigens.

Background: Optimal treatment strategy for the oldest patients with diffuse large B-cell lymphoma (DLBCL) remains controversial, as this group often is precluded from clinical trials, and population-based studies are limited., Methods: All Danish DLBCL-patients ≥75 years diagnosed from 2003 to 2012 were identified, using the Danish National Lymphoma Registry (LYFO). Information regarding baseline characteristics, treatment, comorbidities and outcomes was retrieved from LYFO, the Danish National health registries and medical records. Patients were stratified by age (75-79; 80-84 and 85 + years), comorbidity score and treatment modality (standard treatment [R-CHOP/CHOP-like], less intensive regimens or palliative treatment)., Findings: A total of 1011 patients were included. Standard treatment was initiated in 64%, ranging from 83% among patients aged 75-79 years to 32% among patient aged 85 + years. With standard treatment, median overall survival (OS) estimates were 4·6, 2·6, and 1·9 years for the age groups 75-79, 80-84 and 85+ years. Among patient aged 75-79 and 80-84 years, OS was superior with standard treatment, although high comorbidity scores attenuated this association. Among patients aged 85+ years, survival was not influenced by treatment intensity. Patients ≥80 years had similar OS regardless of intended (R-)CHOP dosing, whereas patients of 75-79 years scheduled for full dose had higher OS. Standard treatment was not associated with increased hospitalisation., Interpretation: Standard treatment is feasible with good outcomes in a large proportion of elderly DLBCL-patients. Planned dose reduction in patients aged ≥80 years had no negative impact on OS., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Intercellular communication via gap junctions has an important role in controlling cell growth and in maintaining tissue homeostasis. Connexin 43 (Cx43; also known as GJA1) is the most abundantly expressed gap junction channel protein in humans and acts as a tumor suppressor in multiple tissue types. Cx43 is often dysregulated at the post-translational level during cancer development, resulting in loss of gap junctions. However, the molecular basis underlying the aberrant regulation of Cx43 in cancer cells has remained elusive. Here, we demonstrate that the oncogenic E3 ubiquitin ligase NEDD4 regulates the Cx43 protein level in HeLa cells, both under basal conditions and in response to protein kinase C activation. Furthermore, overexpression of NEDD4, but not a catalytically inactive form of NEDD4, was found to result in nearly complete loss of gap junctions and increased lysosomal degradation of Cx43 in both HeLa and C33A cervical carcinoma cells. Collectively, the data provide new insights into the molecular basis underlying the regulation of gap junction size and represent the first evidence that an oncogenic E3 ubiquitin ligase promotes loss of gap junctions and Cx43 degradation in human carcinoma cells., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

The B-cell lymphoma-2 (Bcl-2) family of proteins play a crucial role in multiple myeloma (MM), contributing to lacking apoptosis which is a hallmark of the disease. This makes the Bcl-2 proteins interesting targets for therapeutic peptide vaccination. We report a phase I trial of therapeutic vaccination with peptides from the proteins Bcl-2, Bcl-X L and Mcl-1 in patients with relapsed MM. Vaccines were given concomitant with bortezomib. Out of 7 enrolled patients, 4 received the full course of 8 vaccinations. The remaining 3 patients received fewer vaccinations due to progression, clinical decision of lacking effect and development of hypercalcemia, respectively. There were no signs of toxicity other than what was to be expected from bortezomib. Immune responses to the peptides were seen in all 6 patients receiving more than 2 vaccinations. Three patients had increased immune responses after vaccination. Vaccination against Bcl-2 was well tolerated and was able to induce immune responses in patients with relapsed MM., Competing Interests: The authors have no conflicts of interest to declare.

In order to achieve accurate chromosome segregation, eukaryotic cells undergo a dramatic change in morphology to obtain a spherical shape during mitosis. Interphase cells communicate directly with each other by exchanging ions and small molecules via gap junctions, which have important roles in controlling cell growth and differentiation. As cells round up during mitosis, the gap junctional communication between mitotic cells and adjacent interphase cells ceases. Whether mitotic cells use alternative mechanisms for mediating direct cell-cell communication during rounding is currently unknown. Here, we have studied the mechanisms involved in the remodeling of gap junctions during mitosis. We further demonstrate that mitotic cells are able to form actin-based plasma membrane bridges with adjacent cells during rounding. These structures, termed "mitotic nanotubes," were found to be involved in mediating the transport of cytoplasm, including Rab11-positive vesicles, between mitotic cells and adjacent cells. Moreover, a subpool of the gap-junction channel protein connexin43 localized in these intercellular bridges during mitosis. Collectively, the data provide new insights into the mechanisms involved in the remodeling of gap junctions during mitosis and identify actin-based plasma membrane bridges as a novel means of communication between mitotic cells and adjacent cells during rounding.

Purpose: The Region of Southern Denmark (RSD), covering 1.2 of Denmark's 5.6 million inhabitants, established a task force to (1) retrieve literature evidence for the clinical use of positron emission tomography (PET)/CT and provide consequent recommendations and further to (2) compare the actual use of PET/CT in the RSD with these recommendations. This article summarizes the results., Methods: A Work Group appointed a professional Subgroup which made Clinician Groups conduct literature reviews on six selected cancers responsible for 5,768 (62.6 %) of 9,213 PET/CT scans in the RSD in 2012. Rapid Evidence Assessment was applied, using the methodology of systematic reviews with predefined limitations to search PubMed, Embase and the Cochrane Library for articles published in English/Danish/Swedish/Norwegian since 2002. PICO questions were defined, data recorded and quality appraised and rated with regard to strength and evidence level. Consequent recommendations for applications of PET/CT were established. The actual use of PET/CT was compared with these, where grades A and B indicated "established" and "useful" and grades C and D "potentially useful" and "non-recommendable" indications, respectively., Results: Of 11,729 citations, 1,729 were considered for review, and 204 were included. The evidence suggested usefulness of PET/CT in lung, lymphoma, melanoma, head and neck, and colorectal cancers, whereas evidence was sparse in gynaecological cancers. The agreement between actual use of PET/CT and literature-based recommendations was high in the first five mentioned cancers in that 96.2 % of scans were made for grade A or B indications versus only 22.2 % in gynaecological cancers., Conclusion: Evidence-based usefulness was reported in five of six selected cancers; evidence was sparse in the sixth, gynaecological cancers. Actual use of PET/CT agreed well with recommendations.

Background: Magnetic resonance imaging (MRI) makes a significant contribution to diagnose brain injury in premature infants and is a diagnostic procedure that requires the infant to be taken out of the controlled environment established for growth and development. To ensure safe procedures for these vulnerable patients, practical planning and surveillance are paramount., Purpose: This systematic review summarizes and evaluates the literature reporting on practical planning to maintain required safety for premature infants undergoing MRI., Methods: Literature identified through various search strategies was screened, abstracted, appraised, and synthesized through a descriptive analysis. Thirteen research studies, 2 quality improvement projects, and 10 other documents, including practice guidelines, general reviews and articles, a book chapter, and an editorial article, were retained for in-depth review., Conclusions: Various procedures and equipment to ensure the safety of premature infants during MRI have been developed and tested. Although the results are promising and increasingly consistent, our review suggests that more research is needed before conclusive recommendations for the use of magnetic resonance-compatible incubators, the "feed-and-sleep" approach to avoid sedation, or the specific noise-cancelling ear protection for the premature infants' safety during MRI can be established.

Objectives: In this paper we present a contemporary understanding of "nursing informatics" and relate it to applications in three specific contexts, hospitals, community health, and home dwelling, to illustrate achievements that contribute to the overall schema of health informatics., Methods: We identified literature through database searches in MEDLINE, EMBASE, CINAHL, and the Cochrane Library. Database searching was complemented by one author search and hand searches in six relevant journals. The literature review helped in conceptual clarification and elaborate on use that are supported by applications in different settings., Results: Conceptual clarification of nursing data, information and knowledge has been expanded to include wisdom. Information systems and support for nursing practice benefits from conceptual clarification of nursing data, information, knowledge, and wisdom. We introduce three examples of information systems and point out core issues for information integration and practice development., Conclusions: Exploring interplays of data, information, knowledge, and wisdom, nursing informatics takes a practice turn, accommodating to processes of application design and deployment for purposeful use by nurses in different settings. Collaborative efforts will be key to further achievements that support task shifting, mobility, and ubiquitous health care.

Objectives: Thalidomide and bortezomib have been frequently used for second-line therapy in patients with myeloma relapsing after or refractory to initial melphalan-based treatment, but no randomized trials have been published comparing these two treatment alternatives., Methods: Thalidomide- and bortezomib-naïve patients with melphalan refractory myeloma were randomly assigned to low-dose thalidomide + dexamethasone (Thal-Dex) or bortezomib + dexamethasone (Bort-Dex). At progression on either therapy, the patients were offered crossover to the alternative drug combination. An estimated 300 patients would be needed for the trial to detect a 50% difference in median PFS between the treatment arms., Results: After inclusion of 131 patients, the trial was prematurely closed because of low accrual. Sixty-seven patients were randomized to Thal-Dex and 64 to Bort-Dex. Progression-free survival was similar (median, 9.0 months for Thal-Dex and 7.2 for Bort-Dex). Response rate was similar (55% for Thal-Dex and 63% for Bort-Dex), but time to response was shorter (P < 0.05) and the VGPR rate higher (P < 0.01) for Bort-Dex. Time-to-other treatment after crossover was similar (median, 13.2 months for Thal-Dex and 11.2 months for Bort-Dex), as was overall survival (22.8 months for Thal-Dex and 19.0 for Bort-Dex). Venous thromboembolism was seen in seven patients and cerebrovascular events in four patients in the Thal-Dex group. Severe neuropathy, reactivation of herpes virus infections, and mental depression were more frequently observed in the Bort-Dex group. In the quality-of-life analysis, no difference was noted for physical function, pain, and global quality of life. Fatigue and sleep disturbances were significantly more prevalent in the Bort-Dex group., Conclusions: Thalidomide (50-100 mg daily) in combination with dexamethasone seems to have an efficacy comparable with that of bortezomib and dexamethasone in melphalan refractory myeloma. However, the statistical strength of the results in this study is limited by the low number of included patients., (© 2012 John Wiley & Sons A/S.)

Patients with multiple myeloma (MM) suffer from a general impaired immunity comprising deficiencies in humoral responses, T-cell responses as well as dendritic cell (DC) function. Thus, to achieve control of tumour growth through immune therapy constitutes a challenge. Careful evaluation of the immune status in patients with MM seems crucial prior to active immune therapy. We evaluated the proportion of both, DC, Treg cells and myeloid-derived suppressor cells (MDSC) in peripheral blood from patients with MM at diagnosis and in remission as well as patients with monoclonal gammopathy of undetermined significance (MGUS). We found that the proportion of both myeloid (m) DC and plasmacytoid (p) DC in patients at diagnosis was lowered compared to control donors, while only the proportion of pDC in patients in remission and with MGUS was significantly lower than in controls. The proportion of CD4+FOXP3+ Treg cells was increased in patients at diagnosis and not in patients in remission or with MGUS. Also, Treg cells from patients with MM were functionally intact as they were able to inhibit proliferation of both CD4 and CD8 T cells. Finally, we observed an increase in the proportion of CD14+HLA-DR⁻/low MDSC in patients with MM at diagnosis, illustrating that this cell fraction is also distorted in patients with MM. Taken together, our results illustrate that, both mDC, pDC, Treg cells and MDSC are affected in patients with MM underlining the fact that the immune system is dysregulated as a consequence of the disease., (© 2010 The Authors. Scandinavian Journal of Immunology © 2010 Blackwell Publishing Ltd.)

Background: Several laboratories have shown that cells with a memory B-cell phenotype can have the same clonotype as multiple myeloma tumor cells., Design and Methods: The aim of this study was to determine whether some memory B cells have the same genetic alterations as their corresponding multiple myeloma malignant plasma cells. The methodology included sorting multiple myeloma or memory B cells into RNA stabilizing medium for generation of subset-specific polymerase chain reaction complementary DNA libraries from one or 100 cells., Results: Cells with the phenotype of tumor plasma cells (CD38(++)CD19(-)CD45(-/+)CD56(-/+/++)) or memory B cells (CD38(-)/CD19(+)/CD27(+)) were isolated by flow activated cell sorting. In samples from all four patients with multiple myeloma and from two of the three with monoclonal gammopathy of undetermined significance, we identified memory B cells expressing multiple myeloma-specific oncogenes (FGFR3; IGH-MMSET; CCND1 high) dysregulated by an IGH translocation in the respective tumor plasma cells. By contrast, in seven patients with multiple myeloma, each of whom had tumor plasma cells with a K-RAS61 mutation, a total of 32,400 memory B cells were analyzed using a sensitive allele-specific, competitive blocker polymerase chain reaction assay, but no K-RAS mutations were identified., Conclusions: The increased expression of a specific "early" oncogene of multiple myeloma (monoclonal gammopathy of undetermined significance) in some memory B cells suggests that dysregulation of the oncogene occurs in a precursor B-cell that can generate memory B cells and transformed plasma cells. However, if memory B cells lack "late" oncogene (K-RAS) mutations but express the "early" oncogene, they cannot be involved in maintaining the multiple myeloma tumor, but presumably represent a clonotypic remnant that is only partially transformed.

Background: Compared with placebo, prophylactic treatment with bisphosphonates reduces risk of skeletal events in patients with multiple myeloma. However, because of toxicity associated with long-term bisphosphonate treatment, establishing the lowest effective dose is important. This study compared the effect of two doses of pamidronate on health-related quality of life and skeletal morbidity in patients with newly diagnosed multiple myeloma., Methods: This double-blind, randomised, phase 3 trial was undertaken at 37 clinics in Denmark, Norway, and Sweden. Patients with multiple myeloma who were starting antimyeloma treatment were randomly assigned in a 1:1 ratio to receive one of two doses of pamidronate (30 mg or 90 mg) given by intravenous infusion once a month for at least 3 years. Randomisation was done by use of a central, computerised minimisation system. Primary outcome was physical function after 12 months estimated by the European Organisation for Research and Treatment of Cancer (EORTC) QLQ-C30 questionnaire (scale 0-100). All patients who returned questionnaires at 12 months and were still on study treatment were included in the analysis of the primary endpoint. This study is registered with ClinicalTrials.gov, number NCT00376883., Findings: From January, 2001, until August, 2005, 504 patients were randomly assigned to pamidronate 30 mg or 90 mg (252 in each group). 157 patients in the 90 mg group and 156 in the 30 mg group were included in the primary analysis. Mean physical function at 12 months was 66 points (95% CI 62·9-70·0) in the 90 mg group and 68 points (64·6-71·4) in the 30 mg group (95% CI of difference -6·6 to 3·3; p=0·52). Median time to first skeletal-related event in patients who had such an event was 9·2 months (8·1-10·7) in the 90 mg group and 10·2 months (7·3-14·0) in the 30 mg group (p=0·63). In a retrospective analysis, eight patients in the pamidronate 90 mg group developed osteonecrosis of the jaw compared with two patients in the 30 mg group., Interpretation: Monthly infusion of pamidronate 30 mg should be the recommended dose for prevention of bone disease in patients with multiple myeloma., Funding: Nordic Cancer Union and Novartis Healthcare., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

In this double-blind, placebo-controlled study, 363 patients with untreated multiple myeloma were randomized to receive either melphalan-prednisone and thalidomide (MPT) or melphalan-prednisone and placebo (MP). The dose of melphalan was 0.25 mg/kg and prednisone was 100 mg given daily for 4 days every 6 weeks until plateau phase. The dose of thalidomide/placebo was escalated to 400 mg daily until plateau phase and thereafter reduced to 200 mg daily until progression. A total of 357 patients were analyzed. Partial response was 34% and 33%, and very good partial response or better was 23% and 7% in the MPT and MP arms, respectively (P < .001). There was no significant difference in progression-free or overall survival, with median survival being 29 months in the MPT arm and 32 months in the MP arm. Most quality of life outcomes improved equally in both arms, apart from constipation, which was markedly increased in the MPT arm. Constipation, neuropathy, nonneuropathy neurologic toxicity, and skin reactions were significantly more frequent in the MPT arm. The number of thromboembolic events was equal in the 2 treatment arms. In conclusion, MPT had a significant antimyeloma effect, but this did not translate into improved survival. This trial was registered at www.clinicaltrials.gov as #NCT00218855.

Background and Aim: The clinical impact of multiparametric flow cytometry (MFC) in multiple myeloma (MM) is still unclear and under evaluation. Further progress relies on multiparametric profiling of the neoplastic plasma cell (PC) compartment to provide an accurate image of the stage of differentiation. The primary aim of this study was to perform global analysis of CD expression on the PC compartment and subsequently to evaluate the prognostic impact. Secondary aims were to study the diagnostic and predictive impact., Design and Methods: The design included a retrospective analysis of MFC data generated from diagnostic bone marrow (BM) samples of 109 Nordic patients included in clinical trials within NMSG. Whole marrow were analyzed by MFC for identification of end-stage CD45(-) /CD38(++) neoplastic PC and registered the relative numbers of events and mean fluorescence intensity (MFI) staining for CD19, CD20, CD27, CD28, CD38, CD44, CD45, CD56, and isotypes for cluster analysis., Results: The median MFC-PC number was 15%, and the median light microscopy (LM)-PC number was 35%. However, the numbers were significant correlated and the prognostic value with an increased relative risk (95% CI) of 3.1 (1.7-5.5) and 2.9 (1.4-6.2), P < 0.0003 and P < 0.004 of MFC-PC and LM-PC counts, respectively. Unsupervised clustering based on global MFI assessment on PC revealed two clusters based on CD expression profiling. Cluster I with high intensity for CD56, CD38, CD45, right-angle light-scatter signal (SSC), forward-angle light-scatter signal (FSC), and low for CD28, CD19, and a Cluster II, with low intensity of CD56, CD38, CD45, SSC, FSC, and high for CD28, CD19 with a median survival of 39 months and 19 months, respectively (P = 0.02)., Conclusions: The MFC analysis of MM BM samples produces diagnostic, prognostic, and predictive information useful in clinical practice, which will be prospectively validated within the European Myeloma Network (EMN). © 2010 International Clinical Cytometry Society., (Copyright © 2010 International Clinical Cytometry Society.)

Proinflammatory cytokines are suspected to play a role in the pathogenesis of multiple myeloma (MM). Therefore, it is possible that inborn genetic variations leading to a modified expression of these cytokines will influence the outcome for these patients. We investigated 348 MM patients undergoing high-dose melphalan treatment followed by Auto-SCT and examined the influence of single nucleotide polymorphisms (SNPs) in genes involved in the inflammatory response. We found that the polymorphism IL-1beta T-31C significantly influenced overall survival (OS; P=0.02) and that carriers of the variant C-allele had a significantly longer survival than homozygous wild-type allele TT-carriers (relative risk 0.6 (95% CI=0.5-0.9); P=0.008). The polymorphisms IL-6 G-174C, IL-10 C592A, PPARgamma2 Pro(12)Ala, COX-2 A-1195G, COX-2 T8473C and NFKB1 ins/del did not influence the OS in this group of patients. Furthermore, homozygous carriers of the variant allele of IL-1beta T-31C were at 1.37-fold (CI=1.05-1.80) increased risk of MM as compared with population-based controls (P=0.02). Our results indicate that IL-1beta is involved in the pathogenesis of MM.

Multiple myeloma (MM) is an incurable B-cell malignancy characterised by uncontrolled growth and accumulation of malignant plasma cells in the bone marrow. Aberrant expression of CD56 in patients with MM is thought to contribute to a worsened disease course and metastasis. We therefore investigated the regulation of the CD56 promoter in relation to typical clinical factors. We used qPCR and FACS to measure the expression levels of CD56, and potential regulatory factors in patients with MM and related these with MM progression/prognosis. The transcription factors BTBD3, Pax5, RUNX1 and MMSET were positively associated with CD56 expression, as was CYCLIN D1, which is involved in disease progression, anti-apoptosis and proliferation. RUNX1 was negatively associated with the survival of stem-cell transplanted patients. Our findings propose four potential activators of the CD56 promoter and for CD56 to be involved in proliferation and anti-apoptosis, leading to disease progression in MM.

Purpose: To determine the safety, dose-limiting toxicity and maximum tolerated dose (MTD) of the novel hydroxamate histone deacetylase inhibitor belinostat (PXD101) in patients with advanced hematological neoplasms., Patients and Methods: Sequential dose-escalating cohorts of three to six patients with hematological malignancies received belinostat administered as a 30-min i.v. infusion on days 1-5 of a 21-d cycle. Experience from a parallel dose-finding study in patients with solid tumors influenced the selection of the final dose., Results: Sixteen patients received belinostat at one of three dose levels: 600 mg/m(2)/d (three patients), 900 mg/m(2)/d (three patients) and 1000 mg/m(2)/d (10 patients), the dose determined to be the MTD in a phase I solid tumor study [Steele et al. (2008) Clin Cancer Res, 14, 804-10]. The most common treatment-related adverse events (all grades) were nausea (50%), vomiting (31%), fatigue (31%) and flushing (31%). No grade 3 or 4 hematological toxicity compared with baseline occurred except one case of grade 3 lymphopenia. There were two related grade 4 adverse events of renal failure observed. Both events occurred in patients with multiple myeloma and had similar characteristics, i.e. an acute episode of decrease in renal function (pre-existing nephropathy in one patient), with a metabolic profile and decrease in tumor burden consistent with tumor lysis syndrome. No other related grade 4 events were noted. The only related grade 3 events noticed in more than one patient were fatigue and neurological symptoms (one patient had status epilepticus in association with uremia and one patient had paresthesia), all other related grade 3 events occurred in single patients. No cardiac events were noted. No complete or partial remissions were noted in these heavily pre-treated (median of four prior regimens) patients. However, five patients, including two patients with diffuse large-cell lymphoma [including one patient with transformed chronic myelocytic leukaemia (CLL)], two patients with CLL and one patient with multiple myeloma, achieved disease stabilization in of two to nine treatment cycles., Conclusions: Intravenous belinostat at 600, 900 and 1000 mg/m(2)/d is well tolerated by patients with hematological malignancies. The study was carried out in parallel to a similar dose-finding study in patients with solid tumors, in which the MTD was determined to be 1000 mg/m(2)/d days 1-5 in a 21-d cycle. This dose can also be recommended for phase II studies in patients with hematological neoplasms.

Allelic loss at chromosome 9q31-34 is a frequent event in many lymphoproliferative malignancies. Here, we examined DBC1 at 9q33.1 as a potential target in lymphomagenesis. DBC1 is a putative tumor suppressor that has been shown to be involved in the regulation of cell growth and programmed cell death. The methylation status of the DBC1 promoter CpG island was examined by methylation-specific PCR, bisulfite sequencing, and methylation-specific melting curve analysis. DBC1 was hypermethylated in 5 of 5 B-cell-derived lymphoma cell lines, 41 of 42 diffuse large B-cell lymphomas, 24 of 24 follicular lymphomas, 5 of 5 mantle cell lymphomas, 4 of 4 small lymphocytic lymphomas, 1 of 2 lymphoplasmacytoid lymphomas, and in 12 of 12 acute lymphoblastic leukemias, but was unmethylated in 1 case of splenic marginal zone lymphoma, in 12 of 12 multiple myelomas, in 24 of 24 reactive lymph nodes, and in 12 of 12 samples of blood lymphocytes from random donors. DBC1 hypermethylation was associated with transcriptional silencing in lymphoma cell lines, and reexpression of this gene could be induced by treatment with the demethylating agent, 5-aza-2'-deoxycytidine. Our data suggest that hypermethylation of the DBC1 promoter region is a frequent event during the development of lymphoproliferative malignancies, and that DBC1 hypermethylation may serve as a marker for these cancers.

Objectives: In multiple myeloma (MM) YKL-40 is present in the bone marrow microenvironment and is suggested to play a role in remodelling of the extracellular matrix. Here, the association between serum YKL-40 and severity of bone disease in MM is investigated., Methods: Serum YKL-40 was measured in 34 MM patients at diagnosis. Bone disease was assessed by radiography and biochemical markers of bone metabolism. Patients were treated with conventional chemotherapy and followed for up to 30 months., Results: Patients with a serum YKL-40 elevated above the age specific reference range (56%) had a higher total X-ray score (P = 0.003) and higher levels of the markers of bone resorption serum C-terminal telopeptide of collagen type I (P = 0.003), urine pyridinoline (P = 0.04) and urine deoxypyridinoline (P = 0.002), while the levels of urine N-terminal telopeptide of collagen type I (NTX-1) and the markers of bone formation equalled those seen in patients with a normal serum YKL-40. Serum YKL-40 level (beta = 7.6; P = 0.002) and urine NTX-1 (log(2)) (beta = 3.5; P = 0.006) were independent predictors of total X-ray score at diagnosis. Patients with elevated serum YKL-40 at diagnosis had shorter time to first osteolytic event compared to patients with normal serum YKL-40 (12 months vs. not reached; Log rank test: P = 0.03)., Conclusions: Newly diagnosed MM patients with elevated serum concentrations of YKL-40 have more severe bone destruction including increased bone resorptive activity and shorter time to progression of bone disease. At this point, a potential role for YKL-40 in myeloma-related bone disease must be considered.

Osteolytic bone disease (OBD) in multiple myeloma (MM) is caused by interactions between MM cells and the bone marrow microenvironment and is characterized by increased osteoclastic bone resorption and decreased osteoblastic bone formation. Recently, the role of osteoblast inhibition has come into focus, especially the possible role of overexpression of DKK1, an inhibitor of the Wnt signalling pathway. Further, CKS2, PSME2 and DHFR have also been reported as candidate genes for OBD. We studied the gene expression by quantitative reverse transcription polymerase chain reaction of TNFSF11 (RANKL), TNFSF11A (RANK), TNFRSF11B (OPG), CCL3 (MIP1A), CCL4 (MIP1B), PTHR1 (PTHrp), DKK1, CKS2, PSME2 and DHFR in purified, immunophenotypic FACS-sorted plasma cells from 171 newly diagnosed MM patients, 20 patients with monoclonal gammopathy of undetermined significance and 12 controls. The gene expressions of the analysed genes were correlated with radiographically assessed OBD. Only overexpression of DKK1 was correlated to the degree of OBD. Myeloma cells did not express TNFSF11A, TNFSF11, or TNFRSF11B, and very rarely expressed CCL3 and PTHR11. CCL4, CKS2, PSME2 and DHFR were variably expressed, but the expression of these genes showed no correlation with OBD. In contrast, loss of PSME2 expression in MM plasma cells was significantly correlated with OBD.

CD47 and thrombospondin 1 and 2 (TSP1 and TSP2) expression were analysed by real-time reverse transcription polymerase chain reaction in fluorescence-activated cell sorted plasma cells (PCs) from patients at consecutive stages of multiple myeloma (MM) development. 80% of MM patients, but only 39% of patients with monoclonal gammopathy of undetermined significance (MGUS) expressed CD47; median expression level increased 10-fold with progression from MGUS to MM. Elevated TSP1/TSP2 levels occurred in bone marrow cultures from MM patients compared with healthy donors. CD47 and TSP1/TSP2 may have a potential role in the pathophysiology of MM, probably in the interaction between MM PCs and the microenvironment.

Purpose: To evaluate application of MRI and magnetic resonance spectroscopy (MRS) to monitor bone marrow cellularity during pretransplant priming with chemotherapy and hematopoietic growth factor (HGF) administration., Materials and Methods: A total of 10 lymphoma and myeloma patients, in remission following induction therapy and considered eligible for high-dose therapy and autologous stem cell transplantation, were included in the study. MR investigation was scheduled four times: at study entry, and one, two, and four weeks following priming. Priming with cyclophosphamide and recombinant human granulocyte colony-stimulating factor (rhG-CSF) started the day after study entry. MR parameters studied in a region of interest were as follows: bone marrow intensity on short-time inversion-recovery (STIR) turbo spin-echo (TSE; thus STIRTSE) and on T1-weighted TSE (T1TSE) images, T2 value for fat component, T2 value for water component, water/fat ratio (W/F), T1 value for fat component, and T1 value for water component., Results: The results did not support the hypothesis that hematopoietic expansion quantitated and monitored by MR correlates to the level of mobilized progenitor cells., Conclusion: The results indicate that release of stem cells is a more complex phenomenon than hematopoietic expansion and reduction of fat tissue in bone marrow., ((c) 2006 Wiley-Liss, Inc.)

Objectives: A potential role in cancer biology is suggested for YKL-40 (CHI3L1, HC gp-39). The purpose of this study was to evaluate the clinical value of serum YKL-40 (sYKL-40) in multiple myeloma (MM) and to examine YKL-40 expression in malignant plasma cells (MM PCs)., Methods: sYKL-40 was measured by enzyme-linked immunosorbent assay (ELISA) in 82 patients with newly diagnosed MM. YKL-40 expression in immunophenotypically defined plasma cells was investigated by double-labelled immunohistochemistry in 21 MM patients and by real-time reverse transcriptase polymerase chain reaction (RT-PCR) in cDNA archives generated by global RT-PCR in seven controls, 14 patients with monoclonal gammopathy of undetermined significance (MGUS), 45 MM patients, nine patients with extramedullary myeloma (exMM), and seven human myeloma cell lines (HMCLs)., Results: sYKL-40 was elevated above a constructed reference range for healthy controls in 29% of the patients investigated. Patients with elevated sYKL-40 had reduced overall survival and event-free survival when compared to patients with normal sYKL-40, but sYKL-40 level was defeated by beta(2)-microglobulin in the multivariate analyses. Intramedullary MM PCs lacked significant expression of YKL-40, but high levels of YKL-40 expression were seen in extramedullary MM PCs from one exMM patient and in six HMCLs. Further investigations of other bone marrow (BM) cells showed YKL-40 expression in megakaryocytes, neutrophils and adherent cells from long-term BM cultures., Conclusions: In newly diagnosed MM-patients, a sYKL-40 elevated above the reference range predicts a poor clinical outcome, and YKL-40 is expressed by other BM cells than MM PCs. At this point, routine measurements of sYKL-40 are not warranted, but YKL-40 should be considered as a potential player in the pathophysiology of MM.

Background and Objectives: From 1994 to 1997 we conducted a population-based, prospective study on intensive therapy in newly diagnosed symptomatic myeloma patients younger than 60 years, comparing their survival to that of a conventionally treated historic population. Long-term results are presented, including the impact of the degree of response on survival and relapse pattern after transplantation., Design and Methods: The prospective population was formed of 397 patients and the historic population of 313 patients. Both populations were calculated to comprise more than 75% of the expected number of new cases., Results: After a median follow-up of 7 years survival was longer in the prospective population than in the historic one (median 60 versus 39 months; p=0.0002). When comparing only patients eligible for intensive therapy the median survival was 63 versus 44 months (p<0.0001). Attaining a complete response was associated with prolonged event-free survival but not overall survival. The pattern of relapse after transplantation was heterogeneous but could be divided into four major groups; insidious, classical, plasmacytoma form and transformed disease. The median survival after relapse was 29 months. The relapse pattern and time to relapse predicted outcome. Patients relapsing with an insidious or classical form of disease with skeletal events only, or after a long lasting first response were likely to respond well to conventional salvage therapy. In contrast, relapse with multiple symptoms, transformed disease or a short duration of first response implied bad prognosis., Interpretation and Conclusions: The relapse pattern after autologous transplantation is heterogeneous and response to salvage therapy is variable. The degree of response and event-free survival after transplantation are not reliable surrogate markers for survival.

Background: Pre-transplant clinical evaluation of autografting is an important step in predicting post-transplant support, complications and safety. Today, unfavorable outcomes such as early death or graft failure are rare, making them unsuitable for quality assessment of supportive autografting. However, end-points constructed from frequently occurring clinical events may estimate clinically relevant prognostic models., Methods: The present retrospective analysis was based on two consecutive clinical trials in the Nordic area including up to 640 newly diagnosed multiple myeloma patients., Results: In the model, the efficacy (time on antibiotics and use of transfusions) was influenced by pre-transplant variables, including sex, nationality, serum creatinine, hemoglobin, disease stage at diagnosis, response following induction therapy, length of priming and average graft CD34+ cell number per day of harvest. The toxicity end-point (time to blood cell recovery) was influenced by nationality, marrow plasma cell percentage, serum creatinine, M-component isotype, response to induction therapy, length of priming and graft CD34+ cell number. The safety (early disease recurrence or death) was influenced by serum creatinine, hemoglobin, treatment response and CD34+ cell number., Discussion: In conclusion, the model illustrates that intervention strategies in quality assessment of autografting may benefit from probability estimates of graded clinical end-points.

The impact of renal failure on prognosis of multiple myeloma patients treated with high-dose chemotherapy and stem cell support is incompletely studied. A total of 137 patients received high-dose chemotherapy with autologous transplantation at our centre. The patient population was divided into three groups based on their estimated creatinine clearance (Ccr); renal failure defined as Ccr < 60 mL/min: Group A: normal renal function both at diagnosis and at transplant (n = 78), Group B: renal failure at diagnosis but normal renal function at transplant (n = 30), Group C: renal failure both at diagnosis and at transplant (n = 29). There were no differences in the number of stem cells harvested, time to engraftment or response to transplantation between the groups. Ten of the patients in Group C had a normalisation of renal function post-transplant. Significantly longer hospitalisation, increased use of blood products and increased number of infections were seen in Group C compared to Groups A and B. The transplant-related mortality was 17% in Group C compared to 0% and 1% in Groups B and A respectively. Eight patients were on dialysis during transplant and four of these died within the first 100 d post-transplant. Disease response was similar in the three groups. Overall survival was significantly longer in Group A than in Groups B and C. High-dose chemotherapy with autologous transplantation is feasible in MM with renal failure. Whereas patients with moderate renal insufficiency seem to benefit from this treatment, patients in need for dialysis at the time of transplant must be carefully evaluated before proceeding to high-dose chemotherapy., ((c) Blackwell Munksgaard 2005.)

Haematological and infectious toxicity was correlated to renal function in 272 newly diagnosed myeloma patients given standard dose melphalan-prednisone (MP) as initial treatment without dose adjustment for renal impairment. The glomerular filtration rate (GFR) was estimated by calculated creatinine clearance. Haematological toxicity was found to be significantly related to renal dysfunction. Haematological toxicity World Health Organization (WHO) grades 3-4 after the first MP course was seen in 18%, 28% and 36% of patients with a creatinine clearance of >50, 30-50 and <30 ml/min respectively. WHO grades 3-4 infections occurred in 6% and were not significantly related to renal function. We conclude that MP therapy can be used for initial therapy in myeloma patients with renal impairment but suggest that reduction of the melphalan dose should be considered in patients with a GFR of <30 ml/min. As only 2% of our patients had a clearance of < or =10 ml/min no conclusions can be drawn for this subgroup.

Multiple myeloma (MM) is a B cell malignancy characterized by accumulation of plasma cells (PCs) in the bone marrow. Traditional methods for the detection of minimal residual disease (MRD) measure the presence of monoclonal immunoglobulin protein secreted by the malignant PCs. However, changes in the level of MRD in MM may span 6 logs, and methods with a high sensitivity and dynamic range are necessary for quantitating MRD in MM. The two main technologies used in MRD detection are flow cytometry and patient-specific reverse transcription (RT) PCR. Patient-specific RT-PCR has high sensitivity and may be beneficial in monitoring patients receiving allogeneic transplantation. However, for the MRD evaluation of autotransplants, where few patients achieve molecular remission, flow cytometry monitoring seems to be sufficient., (Copyright 2004 S. Karger AG, Basel)

To investigate the frequency and possible biological consequences of c-maf dysregulation, we designed c-maf and IL-4 real-time RT-PCR assays for determination of c-maf and IL-4 mRNA levels. Using the c-maf real-time RT-PCR assay, we tested a panel of 14 B-cell lines, 135 diagnostic bone marrow (BM) samples from patients with multiple myeloma and 10 BM samples from normal donors. In B cell lines and flowsorted CD38++/CD19-/CD56++ myeloma plasma cells (N = 14) the c-maf/GAPDH and IL-4/GAPDH ratios were determined simultaneously using real time RT-PCR. All B cell lines used in the study were characterized by flow cytometry and tested for the presence of Ebstein-Barr virus (EBV). B-cell lines, that were PCR negative for EBV and had a phenotype typical for primary myeloma cells, expressed medium to high levels of c-maf mRNA. However, all EBV PCR positive cell lines, showed a more immature phenotype, lacked expression of aberrant surface markers and contained very low levels of c-maf mRNA. In 4.4% (6/135) of MM patients tested, a c-maf mRNA level comparable to the cell line RPMI 8226 containing at (16:22), translocation was found. In addition, all c-maf positive myeloma cell lines and CD38++/CD19-/CD56++ myeloma plasma cells tested were IL-4 negative. In conclusion, high levels of c-maf mRNA were observed in "true MM cell lines" and 4.4% of MM patients. Further, c-maf dysregulation in myeloma plasma cells did not cause induction of IL-4 transcription.

The t(4:14) translocation affects two potential oncogenes, FGFR3 and MMSET, in multiple myeloma (MM). We investigated the frequency of FGFR3 dysregulation and its prognostic value in MM. FGFR3 mRNA levels were determined in 110 diagnostic bone marrow (BM) samples from MM patients. In addition, selected BM samples were screened for elevated MMSET mRNA levels. 14.5% (16/110) of MM BM samples showed dysregulated FGFR3 expression. Follow-up of 76 MM patients showed no significant difference between FGFR3 dysfunction and survival (P = 0.3) or correlation with known prognostic factors. Further, no linear relation was observed between FGFR3 and MMSET levels.

Objective: Cyclin D1 dysregulation has been found with varying frequencies in multiple myeloma (MM) and has been suggested to be associated with a poor prognosis. The aim of this study was to investigate the frequency of cyclin D1 dysregulation in patients being treated for MM and to test whether cyclin D1 dysregulation is a prognostic factor for MM patients., Methods: To achieve the above aims we designed a highly sensitive and reproducible real-time reverse-transcription polymerase chain reaction (RT-PCR) assay for quantitation of cyclin D1 mRNA. Using this assay, 110 diagnostic bone marrow (BM) samples from patients with MM were screened for cyclin D1 dysfunction., Results: The real-time assay was able to detect the presence of 0.01% cyclin D1 positive cells allowing a safe detection in MM BM samples. In 42% (46/110) of MM BM samples a greater-than-or-equals 3-fold increase in cyclin D1 mRNA was observed compared to the cyclin D1 level in normal BM. In the remaining group of MM patients the cyclin D1 mRNA levels were comparable to normal donors. Follow-up of 76 MM patients showed no significant (P = 0.35) difference in survival between cyclin D1 positive and negative MM patients. In addition, cyclin D1 dysregulation did not correlate with known prognostic factors., Conclusion: The developed real-time RT-PCR assay for detection of cyclin D1 mRNA levels offers a fast and safe screening for cyclin D1 dysfunction. When a large cohort of MM patients was screened, the cyclin D1 gene was found to be frequently dysregulated, but there was no significant correlation to survival or known prognostic parameters.

Background: Cells belonging to the malignant clone are found in the peripheral blood in myeloma patients. In order to minimise the content of tumour cells in the stem cell product it is crucial to perform stem cell harvest at a time when tumour cells in the peripheral blood are at a minimum., Objective: The aim of the study was to compare the mobilisation kinetics of normal CD34+ cells and myeloma plasma cells during mobilisation with either G-CSF alone or high-dose cyclophosphamide (HDCy) plus G-CSF., Design and Methods: Morning blood samples were drawn each day during mobilisation from start of G-CSF or HDCy and to the end of leukapheresis, and were analysed by flow cytometry for content of CD34+ cells and myeloma plasma cells (CD38+ + CD45-). Tumour cells were also estimated by a patient-specific real-time polymerase chain reaction (PCR) method based on the 5' nuclease TaqMan technology., Results: Flow cytometry data from 16 patients showed concomitant mobilisation of CD34+ cells and myeloma plasma cells. Seven patients were mobilised twice; first with G-CSF alone and then with HDCy plus G-CSF. There was no difference between the two mobilisation regimens regarding tumour cell mobilisation kinetics. Real-time PCR was performed in one patient and confirmed the mobilisation of tumour cells at the time when CD34+ blood cells were at a maximum., Conclusions: Tumour cells are mobilised to the peripheral blood at the same time as CD34+ cells in multiple myeloma patients after priming with both G-CSF alone and HDCy in combination with G-CSF.

The tossing of caramels is a traditional part of the celebrations on the last day of school. A case in which a 14-year-old boy suffered a secondary posttraumatic hyphaemia induced by a caramel tossed at the patient's left eye is described. The condition remitted spontaneously without loss of eyesight.

In a population-based study, the Nordic Myeloma Study Group found a survival advantage for high-dose melphalan with autologous blood stem-cell support compared to conventional chemotherapy in myeloma patients under 60 yr of age (risk ratio: 1.62; confidence interval [CI] 1.22-2.15; p = 0.001). A study of health-related quality of life (HRQoL) was integrated in the trial, using the EORTC QLQ-C30 questionnaire. Of the 274 patients receiving intensive therapy 221 (81%) were compared to 113 (94%) of 120 patients receiving conventional melphalan-prednisone treatment. Prior to treatment, there were no statistically significant differences in any HRQoL score between the two groups. One month after the start of induction chemotherapy, the patients on intensive treatment had more sleep disturbance than the control patients. At 6 mo, corresponding to a mean of 52 d after high-dose melphalan, the patients on intensive treatment had moderately lower scores for global QoL and role and social functioning and there was also a significantly higher score for appetite loss. At 12 and 24 mo, the HRQoL was similar to that of the control patients. At 36 mo, there was a trend toward less fatigue, pain, nausea, and appetite loss in the intensive-treatment group. Thus, the 18 mo of prolonged survival seem to be associated with a good health-related quality of life. Despite the moderate HRQoL reduction associated with the early intensive chemotherapy phase, this treatment modality must be regarded as an important step forward in the care of multiple myeloma.

A sufficient number of CD34+ cells in the peripheral blood stem cell product is important to achieve a rapid and sustained engraftment. The purpose of the present work was to study CD34+ cell kinetics during leukapheresis. Blood samples before and after leukapheresis were analysed for CD34+ cells in 205 procedures. The number of CD34+ cells after plus the number of CD34+ cells harvested was 1.5-fold greater than the number available at the beginning of the procedure, indicating recruitment of CD34+ cells during leukapheresis. In a subgroup of 66 procedures, granulocytes and platelets were measured. In contrast to CD34+ cells, these cell fractions were not recruited to the blood stream during leukapheresis. An additional nine patients were studied with serial blood measurements during leukapheresis, showing an initial decline that was followed by an increase in CD34+ cells during leukapheresis. In conclusion, CD34+ cells are recruited to the blood during the leukapheresis procedure in contrast to granulocytes and platelets., (Copyright 2001 Wiley-Liss, Inc.)