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The impending expiry of the patent on a number of leading biologic drugs has led to a surge in the development of ‘biosimilar’ or ‘follow-on’ products. However, in contrast to generic small-molecule medicines, biosimilars are not identical to their reference products. The differences and complexities surrounding both the molecular structure and the manufacturing process for biologics and biosimilars have resulted in a lack of clarity regarding the terms used in different parts of the world to define various aspects of development and utilization such as regulatory approval, pharmacovigilance, interchangeability and treatment-naivety. This makes quantitative evaluation of biosimilars a great challenge to both the scientific community and regulatory agencies.  This manuscript attempts to clarify the terms used and address an important knowledge gap which is currently resulting in an increasing rush to position biosimilars for certain indications and patients in the absence of agreed upon definitions. [ABSTRACT FROM PUBLISHER]

Abstract: Globally, lung cancer remains the most common malignancy and the leading cause of cancer-related death. Whilst resection is a therapeutic option for patients with early stage non-small cell lung cancer (NSCLC), most patients have locally advanced or metastatic disease at diagnosis, the treatment of which still presents a considerable challenge for medical oncologists. Therapeutic cancer vaccines offer a novel approach for the treatment of patients with NSCLC in both the adjuvant and advanced disease settings. Although early attempts to use such technologies were of limited success, increased knowledge of the molecular pathology of tumors, of the immune system in general, and of tumor immunity in particular, has facilitated the production of more sophisticated anticancer vaccines. A number of promising vaccine candidates based on different types of antigenic stimulus have now been evaluated in clinical studies. These include belagenpumatucel-L, a vaccine derived from four genetically modified, irradiated NSCLC cell lines and target protein-specific vaccines designed to induce responses against epidermal growth factor (EGF), melanoma-associated antigen A3 (MAGE-A3) and mucin 1 (MUC1). The purpose of this review is to describe the mode of action of the vaccine candidates that are most advanced in their clinical development for the treatment of NSCLC, and to summarize the most recent data from clinical studies of these vaccines. [Copyright &y& Elsevier]

A large number of human malignancies are associated with decreased numbers of circulating T cells. B-CLL, in this regard, represents an anomaly since there is not only high numbers of circulating B cells, characteristic of the malignancy, but also a massive expansion of both CD4 and CD8 T cells. These T cells for the most part may probably not represent a leukaemia-specific TCR-dependent expansion. On the contrary, these T cells, especially the CD4 subset, might support a “microenvironment” sustaining the growth of the leukaemic B cell clone. Conversely, the leukaemic B cells may produce membrane-bound as well as soluble factors that stimulate the proliferation of these T cells in an antigen independent manner. In addition to these T cells lacking anti-leukaemic reactivity, there exist spontaneously occurring leukaemia-specific T cells recognizing several leukaemia-associated antigens, e.g. the tumour derived idiotype, survivin and telomerase. Both CD4 and CD8 leukaemia-specific T cells have been identified using proliferation and γ-IFN assays. These reactive T cells can lyse autologous tumour cells in an MHC class I and II restricted manner. Spontaneously occurring leukaemia-specific T cells are more frequently noted at an indolent stage rather than in progressive disease. Preliminary results from vaccination trials using whole tumour cell preparations as vaccine have demonstrated that vaccination may induce a leukaemia-specific T cell response, which might be associated with clinical benefits. Extended clinical trials are required to establish the therapeutic effects of vaccination in B-CLL. Studies in our laboratory as well as those of others indicate that whole tumour cell antigen in the form of apoptotic bodies or RNA loaded on to dendritic cells may be a suitable vaccine candidate. Patients with low stage disease may maximally benefit from this form of therapy. [ABSTRACT FROM AUTHOR]

The author comments on the biologic and biosimilars market. Biologics have a large impact on cancer care. The sales volumes of these complex protein drugs have reached more than 1 billion dollars annually. The European Medicine Agency (EMA) defines that when the patent expires on a biologic, biosimilars enter the market.

Explores developments in the biology and treatment of multiple myeloma as of 2000. Incidence rate of the myeloma in the Western world; Factors influencing growth of tumor cells; Importance of bone marrow microenvironment on myeloma disease process.

The ROR1 receptor tyrosine kinase is expressed in embryonic tissues but is absent in normal adult tissues. ROR1 is of importance in oncogenesis and is overexpressed in several cancers, such as NSCLC. In this study, we evaluated ROR1 expression in NSCLC patients (N = 287) and the cytotoxic effects of a small molecule ROR1 inhibitor (KAN0441571C) in NSCLC cell lines. ROR1 expression in tumor cells was more frequent in non-squamous (87%) than in squamous (57%) carcinomas patients, while 21% of neuroendocrine tumors expressed ROR1 (p = 0.0001). A significantly higher proportion of p53 negative patients in the ROR1+ group than in the p53 positive non-squamous NSCLC patients (p = 0.03) was noted. KAN0441571C dephosphorylated ROR1 and induced apoptosis (Annexin V/PI) in a time- and dose-dependent manner in five ROR1+ NSCLC cell lines and was superior compared to erlotinib (EGFR inhibitor). Apoptosis was confirmed by the downregulation of MCL-1 and BCL-2, as well as PARP and caspase 3 cleavage. The non-canonical Wnt pathway was involved. The combination of KAN0441571C and erlotinib showed a synergistic apoptotic effect. KAN0441571C also inhibited proliferative (cell cycle analyses, colony formation assay) and migratory (scratch wound healing assay) functions. Targeting NSCLC cells by a combination of ROR1 and EGFR inhibitors may represent a novel promising approach for the treatment of NSCLC patients. [ABSTRACT FROM AUTHOR]

The receptor tyrosine kinase orphan receptor 1 (ROR1) is absent in most normal adult tissues but overexpressed in various malignancies and is of importance for tumor cell survival, proliferation, and metastasis. In this study, we evaluated the apoptotic effects of a novel small molecule inhibitor of ROR1 (KAN0441571C) as well as venetoclax (BCL-2 inhibitor), bendamustine, idelalisib (PI3Kδ inhibitor), everolimus (mTOR inhibitor), and ibrutinib (BTK inhibitor) alone or in combination in human MCL primary cells and cell lines. ROR1 expression was evaluated by flow cytometry and Western blot (WB). Cytotoxicity was analyzed by MTT and apoptosis by Annexin V/PI staining as well as signaling and apoptotic proteins (WB). ROR1 was expressed both in patient-derived MCL cells and human MCL cell lines. KAN0441571C alone induced significant time- and dose-dependent apoptosis of MCL cells. Apoptosis was accompanied by decreased expression of MCL-1 and BCL-2 and cleavage of PARP and caspase 3. ROR1 was dephosphorylated as well as ROR1-associated signaling pathway molecules, including the non-canonical WNT signaling pathway (PI3Kδ/AKT/mTOR). The combination of KAN0441571C and ibrutinib, venetoclax, idelalisib, everolimus, or bendamustine had a synergistic apoptotic effect and significantly prevented phosphorylation of ROR1-associated signaling molecules as compared to KAN0441571C alone. Our results suggest that targeting ROR1 by a small molecule inhibitor, KAN0441571C, should be further evaluated particularly in combination with other targeting drugs as a new therapeutic approach for MCL. [ABSTRACT FROM AUTHOR]

Recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) is used in immunotherapy for correction of neutropoenia. The optimal dose for activation of immune functions and the pharmacokinetics following repeated administrations is less analysed in depth. In this study, the pharmacokinetics and the effects on haematological functions and antibody-dependent cellular cytotoxicity (ADCC) were analysed in 50 patients with metastatic colorectal carcinoma receiving monoclonal antibody based therapy in combination with Escherichia coli-derived GM-CSF (molgramostim) administered s.c. once daily for 10 days every month over a period of 4 months. Thirty-three patients received a GM-CSF dose of 200–250 μg/m2/day. Seventeen patients received GM-CSF doses varying between 65 and 325 μg/m2/day in the different treatment cycles. Serum GM-CSF concentration was measured (ELISA) before and 3–4 h after (peak serum concentration) GM-CSF administration days 1, 5 and 10. Prior to therapy, GM-CSF was not detectable in serum. Following repeated daily administrations, the peak serum concentration of GM-CSF gradually decreased on days 5 and 10 compared to day 1 ( P < 0.05). During a 10-day treatment cycle, the total number of leukocytes, neutrophils, eosinophils, monocytes and lymphocytes increased. A dose-dependent increment in total white blood cell count and neutrophils was observed. The total numbers of GM-CSF receptor (α-subunit) expressing cells (granulocytes and monocytes) increased significantly during treatment while a transient decline in expression intensity was observed at day 5, suggesting a receptor-mediated removal of GM-CSF as a mechanism for the elimination of GM-CSF from circulation. ADCC of peripheral mononuclear cells was decreased at day 10 compared to baseline. An inverse correlation between the dose and ADCC was noted. The data might indicate that high doses of GM-CSF may have a negative impact on ADCC. [ABSTRACT FROM AUTHOR]

Idiotypic structures expressed on the myeloma immunoglobulin (Ig) protein (M-component) might be regarded as tumor-specific antigens. Naturally occurring, idiotype-specific type I T-cell immunity has been observed preferentially in patients with early stage myeloma. The idiotypic structures on the clonal myeloma B-cells (B lymphocytes and plasma cells) may serve as targets for active immunization. Vaccination using the autologous monoclonal Ig as a vaccine has conferred resistance to tumor cell challenge in murine myeloma. The autologous myeloma Ig protein was used for immunization in patients with progressive stage I and early stage II multiple myeloma. When the idiotype (emulsified in aluminium phosphate) was used alone for immunization, a weak and transient idiotype-specific T-cell response was observed with no clinical effects. In our second series, the idiotype (in alum) was combined with GM-CSF. In all five patients, a specific T-cell response was induced consisting preferentially of MHC class I restricted (CD8 +) T-cells. Ig-specific CD4 + T-cells were also induced. A clinical response (> 50% reduction of the M-component concentration) was observed in one patient. These results indicate that idiotype-specific T-cell immunity may be induced or enhanced by idiotype Ig vaccination in patients with early stage multiple myeloma, in which the tumor load is relatively low and the immune system is functionally less compromized than in patients with chemotherapy-treated, advanced stages of the disease. The use of GM-CSF seems to be mandatory for the frequency and magnitude of the induced T-cell response. The optimal route, schedule and cytokine combination for idiotype immunization remains to be established. [ABSTRACT FROM AUTHOR]

The article focuses on the results of a study which concludes that CD52 induces minimal residual disease-negativity in the bone marrow of poor-prognosis chronic lymphocytic leukaemia patients but is associated with opportunistic infections. It mentions the dual targeting of Bruton tyrosine kinase and trial of combination of ibrutinib and alemtuzumab.

There is a great unmet medical need in pancreatic carcinoma (PC) for novel drugs with other mechanisms of action than existing. PC cells express the onco-fetal RTK ROR1, absent on most normal post-partem cells. ROR1 is involved in proliferation, survival, EMT and metastasis of tumor cells in various malignancies. A small molecule inhibitor (KAN0439834) (530 Da) targeting the TK domain of ROR1 was developed and the activity in ROR1 expressing human PC cell lines (n = 8) evaluated. The effects were compared to a murine mAb against the external part of ROR1, gemcitabine, erlotinib and ibrutinib. KAN0439834 induced significant apoptosis of the tumor cells. EC50 values for KAN0439834 varied between 250–650 nM depending on the cell line. The corresponding values for erlotinib and ibrutinib were 10–40 folds higher. KAN0439834 was much more effective in inducing tumor cell death than the ROR1 mAb although both inhibited ROR1 phosphorylation and downstream non-canonical Wnt pathway molecules. Combination of KAN0439834 with erlotinib or ibrutinib had significant additive effects on tumor cell death. A first-in-class small molecule ROR1 inhibitor (KAN0439834) showed promising in vitro activity against a number of human PC cell lines. Interesting is the additive effects of erlotinib and ibrutinib which warrants further studies as both these agents are in clinical trials for pancreatic carcinoma. [ABSTRACT FROM AUTHOR]

Crosstalk between leukemic cells and the tumor microenvironment is of importance in chronic lymphocytic leukemia (CLL). T cells seem to sustain the survival of CLL cells by various mechanisms. The Krüppel-like family of transcription factors (KLFs) are identified as regulators of proliferation and cell death. In the present study, we analyzed the expression of the wild type (WT) gene KLF6 and the oncogenic splice variant 1 (KLF6–SV1) at the mRNA level in subsets of T cells from CLL patients (n = 29), multiple myeloma patients (n = 6) and normal donors (n = 10). RNA Silencing was used for wtKLF6 and KLF6-SV1. Tumor cell apoptosis was measured. A significant overexpression of wtKLF6 and KLF6-SV1 in T cells of CLL patients compared to normal donors and myeloma patients was noted (p<0.002). Western blot showed that both wtKLF6 and KLF6–SV1 were expressed in purified T cells from CLL patients. KLF6-SV1 siRNA transfection induced a significant down-regulation of KLF6-SV1 in CLL T cells, which lost the capability to sustain the growth of leukemic cells. However, no such a significant effect was seen after wtKLF6 transfection of the autologous T cells. The results suggest that KLF6-SV1 may play a role in the regulation of survival CLL cells. [ABSTRACT FROM AUTHOR]

Purpose: To assess the immunomodulatory and clinical effects of lenalidomide with standard treatment of gemcitabine in patients with advanced pancreatic cancer. Patients and Methods: Patients with advanced pancreatic cancer were treated in first line with lenalidomide orally for 21 days of a 28 days cycle and the standard regimen for gemcitabine. In Part I, which we previously have reported, the dose of lenalidomide was defined (n = 12). In Part II, every other consecutive patient was treated with either lenalidomide (Group A, n = 11) or gemcitabine (Group B, n = 10) during cycle 1. From cycle 2 on, all Part II patients received the combination. Results: A significant decrease in the proliferative response of peripheral blood mononuclear cells and the frequency of DCs were noted in patients at baseline compared to healthy control donors while the frequencies of CD4+ and CD8+ T cells, NK-cells and MDSCs were significantly higher in patients compared to controls. In Group A, a significant increase in the absolute numbers of activated (HLA-DR+) CD4 and CD8 T cells and CD8 effector memory T cells (p<0.01) was noted during treatment. A statistical increment in the absolute numbers of Tregs were seen after cycle 1 (p<0.05). The addition of gemcitabine, reduced most lymphocyte subsets (p<0.05). In Group B, the proportion of lymphocytes remained unchanged during the study period. There was no difference in overall survival, progression free survival and survival rate at one year comparing the two groups. Discussion: Patients with advanced pancreatic carcinoma had impaired immune functions. Lenalidomide augmented T cell reactivities, which were abrogated by gemcitabine. However, addition of lenalidomide to gemcitabine seemed to have no therapeutic impact compared to gemcitabine alone in this non-randomized study. Trial Registration: ClinicalTrials.gov [ABSTRACT FROM AUTHOR]

This phase I-II study explored safety, immunomodulatory and clinical effects of lenalidomide (weeks 1-16) and alemtuzumab (weeks 5-16) in 23 patients with refractory chronic lymphocytic leukemia. Most patients had Rai stage III/IV disease and were heavily pretreated (median 4 prior therapies), and 61% had del(17p)/del(11q). Eleven of 19 evaluable patients (58%) responded, with a median response duration of 12 months (1-29+); time to progression was short in non-responders. Lenalidomide had a narrow therapeutic dose range, 2.5 mg/day was not efficient, and maximum tolerated dose was 5 mg/day. Grade 3-4 neutropenia and thrombocytopenia occurred in 84 and 55%, 30% had febrile neutropenia, and CMV-reactivation requiring valganciclovir occurred in 30% of patients. The frequency of proliferating (Ki67) CD8 T cells was increased at week 4, with further increase in both the CD4 and CD8 subsets ( p < 0.01 and <0.05), which was accompanied by significant upregulation of HLA-DR after addition of alemtuzumab. Antigen-experienced cells increased at week 4 as the frequency of effector memory cells increased in the CD8 subset ( p < 0.003), while effector cells decreased in both the CD8 and CD4 subsets ( p < 0.0001 and p < 0.01). The Th1/Th2 balance was unchanged at week 4 but shifted toward a Th2 profile after combination therapy. At end of treatment, the frequency of Th17 and regulatory T cells was reduced ( p < 0.01), naïve T cells decreased, and effector memory T cells increased ( p < 0.05 and p < 0.01). Granzyme B T cells increased at 30-week follow-up ( p < 0.05). PD-1 expression was unaffected. In conclusion, low-dose lenalidomide and alemtuzumab induced major perturbations of T cells, including increased proliferative activity and cytotoxic potential. [ABSTRACT FROM AUTHOR]

Diffuse large B-cell lymphoma (DLBCL), the most common type of non-Hodgkin's lymphoma (NHL) in adults, accounts for approximately 30-40% of newly diagnosed lymphomas worldwide. Environmental factors, such as viruses and bacteria, may contribute to cancer development through chronic inflammation and the integration of oncogenes, and have previously been indicated in cervical cancer, hepatocellular carcinoma, gastric cancer and lymphoproliferative disorders. In the present study, the presence of microbial agents was analyzed in the lymphoma tissue of patients with activated B-cell like (ABC) DLBCL. The present study compared two groups of patients from geographically varied regions that possess a difference in the prevalence of viral and other microbial agents. The patient populations were from Sweden (a low endemic infectious disease region) and Egypt (a high endemic infectious disease region). A differential expression of several viruses in lymphoma tissues was noted when comparing Swedish and Egyptian patients. JC polyomavirus (JCV) was detected in Swedish and Egyptian patients and, uniquely, the complete hepatitis B virus (HBV) genome was detected only in Egyptian lymphoma patients. None of these viruses were detected in control lymph tissues from Sweden or Egypt. In total, 38% of the Egyptian patients were found to have HBV surface antigens (HBsAgs) in their serum; however, HBsAgs were not found in any of the Swedish patients. The percentage of serum HBsAgs in Egyptian patients with ABC DLBCL was significantly increased compared with the general Egyptian population (P<0.05). The present study may support a notion that viral agents, including JCV and HBV, may be involved in the tumorigenesis of DLBCL in regions of high infectious disease. [ABSTRACT FROM AUTHOR]

Targeted therapies have appeared as new treatment options for several disease types, including cancer and autoimmune disorders. Of several targets, tyrosine kinases (TKs) are among the most promising. Overexpression of TKs provides a target for novel therapeutic agents, including small molecule inhibitors of tyrosine kinases (TKI). Ibrutinib (PCI-32765) is a TKI of Bruton’s tyrosine kinase (Btk), a key kinase of the B-cell receptor signaling pathway that plays a significant role in the proliferation, differentiation and survival of B cells. In addition to inhibitory effects, recent studies have shown that ibrutinib has multiple immunomodulatory effects. It binds covalently to IL-2 inducible tyrosine kinase (Itk) in T lymphocytes and suppresses the survival of T-helper (Th) 2 cells. This changes the balance of Th1/Th2 cells toward Th1 subset, which are the main immune cells targeting tumor cells. The dual activity of ibrutinib has paid a great attention and several studies are evaluating the anti-tumor and immunomodulatory effects in cancer, autoimmune disorders and infectious diseases. In this article we review the inhibitory and immunomodulatory effects of ibrutinib in B-cell malignancies, autoimmune diseases and infections, as well as the communication between the Ror1 receptor tyrosine kinase and BCR and effects of ibrutinib on this crosstalk. [ABSTRACT FROM AUTHOR]

Background: ROR1 is a receptor tyrosine kinase expressed in chronic lymphocytic leukemia (CLL) and several other malignancies but absent in most adult normal tissues. ROR1 is considered an onco-fetal antigen. In the present study we analysed spontaneous humoral and cellular immunity against ROR1 in CLL patients. Materials and Methods: Antibodies against ROR1 were analysed in 23 patients and 20 healthy donors by ELISA and Western blot. Purified serum IgG from patients was tested for cytotoxicity against CLL cells using the MTT viability assay. A cellular immune response against ROR1 derived HLA-A2 restricted 9 aa and 16 aa long peptides were analysed using peptide loaded dendritic cells co-cultured with autologous T cells from CLL patients (n = 9) and healthy donors (n = 6). IFN-γ, IL-5 and IL-17A-secreting T cells were assessed by ELISPOT and a proliferative response using a H3-thymidine incorporation assay. Results: The majority of CLL patients had antibodies against ROR1. Significantly higher titers of anti-ROR1 antibodies were noted in patients with non-progressive as compared to progressive disease. The extracellular membrane-close ROR1 KNG domain seemed to be an immunodominant epitope. Ten patients with high titers of anti-ROR1 binding antibodies were tested for cytotoxicity. Five of those had cytotoxic anti-ROR1 antibodies against CLL cells. ROR1-specific IFN-γ and IL-17A producing T cells could be detected in CLL patients, preferentially in non-progressive as compared to patients with progressive disease (p<0.05). Conclusion: ROR1 seemed to spontaneously induce a humoral as well as a T cell response in CLL patients. The data support the notion that ROR1 might be a specific neo-antigen and may serve as a target for immunotherapy. [ABSTRACT FROM AUTHOR]

The article discusses the role of phosphoinositide 3-kinase (PI3K)/AKT/ mechanistic target of rapamycin (mTOR) pathway in direct apoptosis of chronic lymphocytic leukemia (CLL) cells induced by ROR1 monoclonal antibodies. Topics discussed include regulation of growth of malignant cells, tumour antigens with a specific expression in CLL, and functions of the receptor tyrosine kinases.

Targeted cancer therapies have emerged as new treatment options for various cancer types. Among targets, receptor tyrosine kinases (RTKs) are among the most promising. ROR1 is a transmembrane RTK of importance during the normal embryogenesis for the central nervous system, heart, lung and skeletal systems, but is not expressed in normal adult tissues. However, ROR1 is overexpressed in several human malignancies and may act as a survival factor for tumor cells. Its unique expression by malignant cells may provide a target for novel therapeutics including monoclonal antibodies (mAbs) and small molecule inhibitors of tyrosine kinases (TKI) for the treatment of cancer. Promising preclinical results have been reported in e.g. chronic lymphocytic leukemia, pancreatic carcinoma, lung and breast cancer. ROR1 might also be an interesting oncofetal antigen for active immunotherapy. In this review, we provide an overview of the ROR1 structure and functions in cancer and highlight emerging therapeutic options of interest for targeting ROR1 in tumor therapy. [ABSTRACT FROM AUTHOR]

Objective: Epithelial cell adhesion molecule (EpCAM) has experienced a renaissance lately as a binding site for targeted therapy as well as a prognostic marker in epithelial malignancies. Aim of this study was to study EpCAM as a potential prognostic marker in epithelial ovarian cancer (EOC). Methods: EpCAM expression was assessed by immunohistochemistry on paraffin-embedded primary EOC-tissue samples. EpCAM overexpression was defined as an expression of EpCAM of 76% to 100%. Tissue samples and clinical data were systematically collected within the international and multicenter "Tumorbank Ovarian Cancer" network. Results: Seventy-four patients, diagnosed with EOC between 1994 and 2009, were included in the study (median age, 56 years; range, 31 to 86 years). The majority of the patients (81.1%) presented with an advanced stage International Federation of Gynecology and Obstetrics (FIGO) III/IV disease. Histology was of the serous type in 41 patients (55.4%), endometrioid in 19 (25.6%), and mucinous in 14 (19%). EpCAM was overexpressed in 87.7%. Serous tumors overexpressed EpCAM significantly more often than mucinous tumors (87.8% vs. 78.6%, p=0.045); while no significant difference was noted between the other histological subgroups. EpCAM overexpression was significantly associated with a better progression free survival and higher response rates to platinum based chemotherapy (p=0.040 and p=0.048, respectively). EpCAM was identified as an independent prognostic marker for overall survival (p=0.022). Conclusion: Our data indicate a significant association of EpCAM overexpression with a more favorable survival in EOC-patients. Serous cancers showed a significant EpCAM overexpression compared to mucinous types. Larger multicenter analyses are warranted to confirm these findings. [ABSTRACT FROM AUTHOR]

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease, and infectious agents are suspected to be involved in the tumorigenesis of DLBCL. MicroRNAs (miRNAs) are non-coding RNAs modulating protein expression. We compared miRNA expression profiles in lymph node tissues of patients with DLBCL of the activated B-cell like (ABC) type from two geographical areas with different background exposures, Sweden and Egypt. We showed previously that DLBCL tissues of the ABC-type in Swedish patients had a higher expression of signal transducer and activator of transcription 3 (STAT3) compared to Egyptian patients. Here, we analyzed the involvement of miRNAs in STAT3 regulation. miR-1234 was significantly up-regulated in Egyptian patients with DLBCL compared to Swedish patients ( p < 0.03). The miR-1234 expression level correlated inversely with the expression of STAT3. The Stat3 protein was down-regulated in cells transfected with miR-1234, suggesting that STAT3 might be a potential target for miR-1234. miR-1234 and STAT3 might be involved in the tumorigenesis of DLBCL of ABC type and possibly associated with environmental background exposure. [ABSTRACT FROM AUTHOR]

Telomerase activity and telomere length (TL) are prognostic markers in chronic lymphocytic leukemia (CLL). The rate-limiting component of telomerase is human telomerase reverse transcriptase (hTERT), for which multiple transcripts exist. Two splicing sites, α and β, have been described that generate deleted transcripts. Only the full-length (FL; α+β+) transcript translates into a functional protein. The aim of this work was to characterize hTERT splice variants in CLL in relation to disease activity, clinical stage, immunoglobulin heavy chain variable (IGHV) genes mutational status, and TL. Real-time polymerase chain reaction assays were validated for quantification of the hTERT transcripts with either α deletion (del-α; α−β+), β deletion (del-β; α+β−) or both α and β deletions (del-αβ; α−β−). The splice variant expression pattern was studied in 97 patients with CLL, 6 healthy control subjects, and one CD34+ cell sample. TL was assessed with real-time polymerase chain reaction in 71 of 97 samples. Thirty-two percent of the cases did not express any of the splice variants. Average FL expression was 5.5-fold higher in IGHV-unmutated (n = 35) compared with mutated (n = 59) patients (p < 0.0001). FL levels correlated directly with the percentage of IGHV homology (r = 0.34; p = 0.0007) and inversely with TL (r = −0.44; p = 0.0001). Overall, FL expression correlated significantly with that of the other splice variants. All transcripts were more frequently expressed in progressive compared with nonprogressive patients (p < 0.0001 for FL and del-α; p = 0.01 for del-β; and p = 0.006 for del-αβ). This study provides a detailed insight into the hTERT transcript pattern in CLL, highlighting the necessity of subgrouping patients according to IGHV mutation status when analyzing hTERT expression. [Copyright &y& Elsevier]

Proline/arginine-rich end leucine-rich repeat protein (PRELP) belongs to the small leucine-rich proteoglycan (SLRP) family, normally expressed in extracellular matrix of collagen-rich tissues. We have previously reported on another SLRP, fibromodulin (FMOD) in patients with chronic lymphocytic leukemia (CLL). PRELP is structurally similar to FMOD with adjacent localization on chromosome 1 (1q32.1). As cluster-upregulation of genes may occur in malignancies, the aim of our study was to analyze PRELP expression in CLL. PRELP was expressed (RT-PCR) in all CLL patients (30/30), as well as in some patients with mantle cell lymphoma (3/5), but not in healthy donor leukocytes (0/20) or tumor samples from other hematological malignancies (0/35). PRELP was also detected in CLL cell-lines (4/4) but not in cell-lines from other hematological tumors (0/9). PRELP protein was detected in all CLL samples but not in normal leukocytes. Deglycosylation experiments revealed a CLL-unique 38 kDa core protein, with an intact signal peptide. This 38 kDa protein was, in contrast to the normal 55 kDa size, not detected in serum which, in combination with the uncleaved signal peptide, suggests cellular retention. The unique expression of a 38 kDa PRELP in CLL cells may suggest involvement in the pathobiology of CLL and merits further studies. [ABSTRACT FROM AUTHOR]

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease with an association with inflammation and viral infections. We hypothesize that environmental factors may be involved in the pathogenesis of DLBCL. In this study, we compared gene expression profiles of lymph node tissues from patients with DLBCL from two different geographical areas with diverse environmental exposures. Specimens from Egyptian and Swedish patients with DLBCL as well as controls were studied. Gene expression analysis using microarray and quantitative polymerase chain reaction demonstrated significantly higher expression of signal transducer and activator of transcription 3 (STAT3) in Swedish as compared to Egyptian patients and control materials from both countries. This was confirmed at protein level using confocal microscopy. The receptor tyrosine kinase ROR1, a 'survival factor' for malignant cells, was overexpressed and significantly related to the STAT3 expression pattern. The difference in the expression of genes involved in inflammatory responses and in the tumorigenic process of DLBCL might relate to infectious agents and/or other environmental exposures. [ABSTRACT FROM AUTHOR]

The receptor tyrosine kinase ROR1 has been shown to be overexpressed in chronic lymphocytic leukemia (CLL). The aim of this study was to further characterize the expression of ROR1 and the other member of the ROR family, ROR2, in other lymphoid and myeloid malignancies. Normal white blood cells and reactive lymph nodes were negative for ROR1 and ROR2. A significantly high and uniform surface expression of ROR1 was found in CLL/hairy cell leukemia (HCL) compared to mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), myelomas, acute lymphoblastic leukemia (ALL) and myeloid leukemias ( p = 0.02 to < 0.001). The lowest proportion of ROR1+ cells was seen in FL, whereas CLL, HCL and CML had significantly higher numbers of ROR1+ cells. Longitudinal follow-up of individual patients with CLL revealed that ROR1+ cells remained stable over time in non-progressive patients, but increased when the disease progressed ( p < 0.05). Thus, a variable staining pattern of ROR1 ranging from very high (CLL, HCL) and high (CML) to intermediate (myeloma and DLBCL) or low (FL) was noted. ROR2 was not detected in hematological malignancies. [ABSTRACT FROM AUTHOR]

T cells may have a role in sustaining the leukemic clone in chronic lymphocytic leukemia (CLL). In this study, we have examined the ability of T cells from CLL patients to support the survival of the leukemic B cells in vitro. Additionally, we compared global gene expression of T cells from indolent CLL patients with healthy individuals and multiple myeloma (MM) patients. Apoptosis of purified leukemic B cells was inhibited in vitro when co-cultured with increasing numbers of autologous T cells ( p < 0.01) but not autologous B and T cells of normal donors. The anti-apoptotic effect exceeded that of the anti-apoptotic cytokine IL-4 ( p = 0.002) and was greater with CD8+ cells ( p = 0.02) than with CD4+ cells ( p = 0.05). The effect was depended mainly on cell-cell contact although a significant effect was also observed in transwell experiments ( p = 0.05). About 356 genes involved in different cellular pathways were deregulated in T cells of CLL patients compared to healthy individuals and MM patients. The results of gene expression profiling were verified for 6 genes (CCL4, CCL5 (RANTES), XCL1, XCL2, KLF6, and TRAF1) using qRT-PCR and immunoblotting. Our results demonstrate that CLL-derived T cells can prevent apoptosis of leukemic B cells and have altered expression of genes that may facilitate the survival of the leukemic clone. [ABSTRACT FROM AUTHOR]

Purpose: Previous clinical studies have indicated that natural IgM antibodies have the ability to induce apoptosis of tumor cells but IgE and IgA may also mediate tumor cell killing (in addition to IgG). The aim of the study was to analyse induction of IgM, IgA and IgE antibodies in patients vaccinated with the tumor associated antigen CEA. Methods: Twenty-four resected CRC patients without macroscopic disease were immunized seven times with CEA ± GM-CSF. Four different dose schedules were used over a 12-month period. IgM, IgA and IgE antibody responses against recombinant CEA were determined by ELISA. Patients were monitored immunologically for 36 months and clinically for 147 months. Results: GM-CSF significantly augmented the anti-CEA response for all three antibody classes. Low dose of CEA tended to induce a higher IgM, IgA or IgE anti-CEA antibody response than higher. Anti-CEA IgA antibodies could lyse CEA positive tumor cells in antibody dependent cellular cytotoxicity (ADCC) as well as in complement dependent cytotoxicity (CDC). A significant correlation between survival and high IgA anti-CEA titers was noted ( p = 0.02) irrespective of GM-CSF treatment. Conclusions: The observation that IgA anti-CEA antibodies were cytotoxic and associated with improved survival might indicate that also these antibodies may exert a clinical anti-tumor effect. [ABSTRACT FROM AUTHOR]

We previously demonstrated that autologous dendritic cells that have endocytosed apoptotic bodies of chronic lymphocytic leukemia (CLL) cells (Apo-DC) can stimulate antileukemic T cell responses in vitro. In this phase I study, we vaccinated 15 asymptomatic CLL patients at five time points with Apo-DC administered intradermally either alone (cohort I), or in combination with subcutaneous granulocyte-macrophage-colony-stimulating-factor (GM-CSF) (cohort II) or with GM-CSF and intravenous low-dose cyclophosphamide (cohort III). Aim of the study was to evaluate the safety and immunogenicity of Apo-DC alone or in combination with GM-CSF and low-dose cyclophosphamide in CLL patients. All patients completed the vaccination schedule without dose-limiting toxicity. No objective clinical responses were seen. Vaccine-induced leukemia-specific immune responses were evaluated by IFN-γ ELISpot and proliferation assays over a 52 weeks observation period and immune response criteria were defined. According to these criteria, 10/15 patients were defined as immune responders. The frequency of immune-responding patients was higher in cohorts II (3/5) and III (5/5) than in cohort I (2/5). In order to further characterize the induced immune response, estimation of secreted cytokines and CD107-degranulation assay were performed. Clustering of T and CLL cells was observed in CD107-degranulation assay and visualized by confocal microscopy. Additionally, assessment of regulatory T cells (T) revealed their significantly lower frequencies in immune responders versus non-responders ( P < 0.0001). Cyclophosphamide did not reduce T frequency. In conclusion, vaccination with Apo-DC + GM-CSF and cyclophosphamide was safe and elicited anti-CLL immune responses that correlated inversely with T levels. Lack of clinical responses highlights the necessity to develop more potent vaccine strategies in B cell malignancies. [ABSTRACT FROM AUTHOR]

We have previously demonstrated that ROR1 and FMOD (fibromodulin) are two genes upregulated in chronic lymphocytic leukaemia (CLL) cells compared to normal blood B cells. In this study, siRNAs were used to specifically silence ROR1 and FMOD expression in CLL cells, healthy B cells and human fibroblast cell lines. siRNA treatment induced a specific reduction (75-95%) in FMOD and ROR1 mRNA. Western blot analysis with specific antibodies for FMOD and ROR1 demonstrated that the proteins were significantly downregulated 48 h after siRNA treatment. Silencing of FMOD and ROR1 resulted in statistically significant ( P ≤ 0·05-0·001) apoptosis of CLL cells but not of B cells from normal donors. Human fibroblast cell lines treated with FMOD and ROR1 siRNA did not undergo apoptosis. This is the first report demonstrating that ROR1 and FMOD may be involved in the survival of CLL cells. ROR1 in particular is further explored as potential target for therapy in CLL. [ABSTRACT FROM AUTHOR]

Immunotherapy is being increasingly utilized for adjuvant treatment for breast cancer (BC). We have previously described immune functions during primary therapy for BC. The present study describes immune recovery patterns during long-term, unmaintained follow-up after completion of adjuvant therapy.A group of patients with primary BC had been treated with adjuvant radio-chemotherapy (RT + CT) 5-fluorouracil, epirubicin and cyclophosphamide (FEC) ( n = 21) and another group with radiotherapy (RT) ( n = 20) alone. Immunological testing of NK and T-cell functions was performed initially at the end of adjuvant treatment and repeated after 2, 6 and 12 months. NK cell cytotoxicity was significantly higher ( P < 0.05) at all time-points in patients than in age-matched controls and did not differ between the two treatments groups during one year observation. In contrast, lower numbers of CD4 T-cells and lower expression of CD28 on T-cells was observed particularly in RT + CT patients and did not normalize during the observation period. The numbers of Treg cells (CD4+CD25high) were low in the RT + CT group during follow-up, as well as expression of TCRξ, Zap70, p56lck, P59fyn and PI3 k in CD4+ cells. In contrast, expression of intracellular cytokines (IFN-γ, IL-2, IL-4) in CD4 and CD8 T cells were significantly higher in RT + CT patients than in the RT group and the difference increased during follow-up. In conclusion, NK-cell cytotoxicity increased during unmaintained long-term follow-up whereas CD4 and regulatory T cells as well as signal transduction molecules remained low following adjuvant radio-chemotherapy. [ABSTRACT FROM AUTHOR]

Problem Recurrent spontaneous abortion (RSA) is a relatively common disorder, the underlying causes of which are thought to be immunological in most cases. Method of study Expression profile and clonality pattern of T-cell receptor beta variable (TCRBV) genes in endometrium and blood of patients with RSA were investigated by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) using BV gene-specific primers. Relative expression of each BV family was determined and clonal expansion of the over-expressed genes was assessed by analysis of CDR3 length polymorphism. Results Compared to blood, relative expression of four TCRBV genes was significantly higher in the endometrium of RSA group. Over-expressed genes, except for TCRBV3, all had restricted and oligoclonal patterns of expression in the endometrium. Conclusion Endometrial T cells have a skewed TCRBV repertoire with restricted transcript heterogeneity, which is shared by both groups and minor variations observed in this pattern in RSA patients may reflect more recent and/or repeated exposure to nominal antigens or superantigens. [ABSTRACT FROM AUTHOR]

Goal Of Work: To study the effect of support intervention on immune function in breast cancer patients.Materials and Methods: Breast cancer patients from an ongoing prospective randomised quality-of -life study were chosen for assaying immune functions in relation to a support-group intervention program running on a residential basis. Twenty-one women received adjuvant-combined radio-chemotherapy (CT-RT) and 20 women radiotherapy (RT). Eleven CT-RT and ten RT patients were randomised to support-group intervention, the rest served as controls. Immune tests for NK cells and NK-cell cytotoxicity, as well as lymphocyte subpopulations and response to antigen were performed before intervention, 2, 6, and 12 months later, in parallel to controls and healthy volunteers (n = 11). Depression, anxiety and fatigue were evaluated by the Hospital Anxiety and Depression (HAD) and the Norwegian Fatigue questionnaire. The density of NK cell receptors and in vitro quantitation of functional NK cytotoxicity against K562 cell line were evaluated. Four-colour flow cytometry was used to detect signal transduction molecules and cytokine expression. T-cell proliferate response to purified protein derivate (PPD) antigen was evaluated.Results: No significant immune effect of support intervention could be found. The immune variables were severely disarranged compared to healthy volunteers but showed a statistically significant improvement over time. The majority of patients suffered from fatigue but had low scores for depression and anxiety.Conclusion: No effect on immune parameters could be detected from support intervention. The long-lasting immune suppression might override a putative effect of the intervention. Low depression scores may contribute to the absence of a detectable effect. [ABSTRACT FROM AUTHOR]

Objectives: To elucidate long-term effects of idiotype (Id) vaccination on Id-specific T cells of multiple myeloma (MM) patients and compare Id-specific T-cell responses of peripheral blood with those of bone marrow (BM). Materials and methods: Id-specific T-cell responses of peripheral blood mononuclear cells (PBMC) were compared with those of BM mononuclear cells (BMMC) in 10 MM patients vaccinated with the Id protein at a median time of 41 months since the last immunization. The PBMC responses at late follow-up were also compared with those during active immunization. The responses were assessed by a proliferation assay, enzyme-linked immunospot (ELISPOT) (γ-interferon), cytometric bead array (CBA) for secreted cytokines and quantitative real-time polymerase chain reaction (QRT-PCR) for cytokine gene expression. Results: At the late testing time, an Id-specific response was detected in PBMC of five patients (ELISPOT, CBA, QRT-PCR) and in BMMC of four patients (CBA, QRT-PCR). A response in both compartments was noted only in three patients. The cytokines gene profile was consistent with a predominance of Th2 cells [interleukin (IL)-4, IL-5, IL-10]. Comparison of the Id-specific responses of PBMC during active immunization with those at the late follow-up showed that the frequency and magnitude of the responses had decreased significantly by time (proliferation/ELISPOT) ( P < 0.02) and shifted at the gene level from a Th1 to a Th2 profile ( P < 0.05). Conclusion: Id-specific T cells may decline overtime and shift toward a Th2 response and may be found at a similar frequency of patients in blood and BM. [ABSTRACT FROM AUTHOR]

Objective: Human telomerase reverse transcriptase (hTERT) is the catalytic subunit of telomerase. In B-cell chronic lymphocytic leukemia (B-CLL), telomerase activity is increased in about 75% of patients. The aim of this study was to analyze whether B-CLL patients with telomerase-positive leukemic cells had naturally occurring, telomerase-specific T cells that might be utilized for immune-mediated lysis of autologous tumor cells. Methods: Spontaneous T-cell immunity and cytotoxicity against hTERT was explored in B-CLL. Nineteen of 25 B-CLL patients (76%) expressed hTERT (reverse transcriptase polymerase chain reaction) and 10 were selected for specific T-cell analysis against hTERT. Results: The stimulation index (SI) of T cells from seven telomerase-positive patients stimulated with a 16aa hTERT peptide (611–626) loaded onto dendritic cells (DC) was 33.9 ± 15.4 (mean SI ± standard error of mean) and 13.2 ± 5.6 against a Ras control peptide (p = 0.05), whereas the corresponding SI values for three telomerase-negative patients were 5.3 ± 5.3 against the hTERT 611-626 peptide and 10.3 ± 6.5 against the Ras peptide, respectively; and for three healthy controls, 5.4 ± 0.9 against the hTERT 611-626 peptide and 4.5 ± 1.0 against the Ras peptide (both not significant). Blocking experiments revealed that the specific responses were major histocompatibility complex (MHC) class I and MHC class II restricted. DC pulsed with the hTERT-peptide generated MHC class I-restricted, hTERT-specific cytotoxic T lymphocytes in six of seven telomerase-positive patients; mean cytotoxicity of hTERT-stimulated T cells was 49.8% ± 9.3% vs 13.1 ± 2.9% for Ras-stimulated T cells (p < 0.05). In three of three telomerase-negative patients, no hTERT-specific cytotoxic T lymphocytes could be expanded. Conclusion: Telomerase-positive B-CLL patients have spontaneously occurring cytotoxic hTERT-specific T cells. This antigen might be explored as a therapeutic vaccine in B-CLL. [Copyright &y& Elsevier]

In this pilot study, T-cell receptor B-variable (TCR-BV) gene usage in CD4 and CD8 T cells was assessed, by real-time polymerase chain reaction, as well as complementarity-determining region 3 (CDR3)-length polymorphism, before and after therapy in five patients with B-cell chronic lymphocytic leukaemia who received alemtuzumab (anti-CD52 monoclonal antibody) as first-line therapy. A decline in expression of most BV family genes in both CD4 and CD8 T cells was observed after alemtuzumab treatment, which was followed by a gradual increase in most BV families during long-term follow-up. After treatment, CDR3-length polymorphism showed an even more restricted pattern in CD4 T cells compared with pretreatment, with a shift towards a monoclonal/oligoclonal pattern. The clonally restricted pattern was significantly reduced in CD4 ( P < 0·01) but not in CD8 T cells. This was followed by a gradual increase in the number of peaks within the CDR3 region of the different TCR-BV families, i.e. a polyclonal repertoire, during long-term follow-up. A restricted CDR3 pattern became even more restricted after treatment, but normalised during unmaintained follow-up. These results indicate that perturbations in the T-cell alterations following alemtuzumab are complex and include not only changes in CD4/CD8 T-cell numbers but also a highly restricted T-cell repertoire especially in CD4 T cells. [ABSTRACT FROM AUTHOR]

Fludarabine and alemtuzumab are routinely used for treatment of B-cell chronic lymphocytic leukemia (B-CLL). The present study aimed to compare the expression of signaling molecules and cytokine production by T cells of B-CLL patients in long-term unmaintained remission/plateau phase following fludarabine or alemtuzumab treatment with that of indolent/untreated B-CLL patients and healthy donors. The frequency and intensity of TCR-CD3ζ chain, p56lck, p59fyn, ZAP-70, PI3-kinase and interferon (IFN)-γ/interleukin (IL)-4 production in CD4 and CD8 T cells was examined by flow cytometry. T-cell function was assessed by stimulation with purified protein derivative (PPD) and phytohemagglutinin (PHA). Despite a reduction in number, the expression of IFN-γ/IL-4 in T-cells in patients was significantly higher than in healthy donors. The intensity of most signaling molecules in treated patients was relatively unaffected vs. healthy donors but lower than untreated-indolent patients. However, the total number of T cells which expressed each of the signaling molecules was decreased in patients, with no difference between fludarabine- and alemtuzumab-treated patients. The T-cell response to PHA but not PPD was reduced in treated patients. The results suggest that, despite some alterations in signaling molecules and a reduction in T-cell number, overall T-cell functions may be relatively well preserved long-term after treatment with fludarabine and alemtuzumab. [ABSTRACT FROM AUTHOR]

The tumour-associated antigen, Ep-CAM, is over-expressed in colorectal carcinoma (CRC). In the present study, a recombinant Ep-CAM protein or a human anti-idiotypic antibody (anti-Id) mimicking Ep-CAM, either alone or in combination, was used for vaccination of CRC patients (n=9). GM-CSF was given as an adjuvant cytokine. A cellular immune response was assessed by measuring anti-Ep-CAM lymphoproliferation, IFN-? production (ELISPOT) and by analysing the TCR BV gene usage within the CD4+ and CD8+ T-cell subsets followed by CDR3 fragment analysis. A proliferative and/or IFN-? T-cell response was induced against the Ep-CAM protein in eight out of nine patients, and against Ep-CAM-derived peptides in nine out of nine patients. Analysis of the TCR BV gene usage showed a significantly higher usage of BV12 family in CD4+ T cells of patients both before and after immunisation than in those of healthy control donors (p<0.05). In the CD8+ T-cell subset, a significant (p<0.05) increase in the BV19 usage was noted in patients after immunisation. In individual patients, a number of TCR BV gene families in both CD4+ and CD8+ T cells were over-expressed mainly in post-immunisation samples. Analysis of the CDR3 length polymorphism revealed a higher degree of clonality in post-immunisation samples than in pre-immunisation samples. In vitro stimulation with Ep-CAM protein confirmed the expansion of anti-Ep-CAM T-cell clones. The results indicate that immunisation with the Ep-CAM protein and/or anti-Id entails the induction of an anti-Ep-CAM T-cell response in CRC patients, and suggest that BV19+ CD8+ T cells might be involved in a vaccine-induced immune response. [ABSTRACT FROM AUTHOR]

In susceptible mice, mercuric chloride induces a systemic autoimmune response that is characterized by elevated serum levels of immunoglobulin G1 (IgG1) and immunoglobulin E (IgE), production of anti-nucleolar antibodies (ANolAs) and the formation of renal IgG deposits. We have previously shown that mercury can also enhance immune/autoimmune responses in mouse strains genetically prone to develop spontaneous autoimmune disease. Here, we investigated whether mercury can enhance the severity of murine collagen-induced arthritis (CIA), an inducible (acquired) autoimmune disease that cannot be induced by mercury itself. While mercury administered prior to the induction phase of CIA exerted little, if any, influence, administration of mercury during the induction phase and following onset aggravated the symptoms of this disease and increased the serum levels of IgE and IgG2a antibodies directed against collagen type II (CII). Furthermore, while animals injected with mercury alone exhibited a significant decrease in the ratio of the levels of interferon-γ (IFN-γ) to interleukin-4 (IL-4) mRNA in their spleens, this ratio was increased in mice with CIA, with or without administration of mercury. Finally, the production of anti-nuclear antibodies, a hallmark of autoimmunity in response to mercury, was observed in all mice with CIA treated with this heavy metal. Our findings suggest that exposure to mercury during the development of CIA may influence immunological factors in such a way as to synergistically promote disease development. [ABSTRACT FROM AUTHOR]

A baculovirus-produced recombinant CEA (rCEA) protein comprising the extracellular region was used for vaccination of CRC patients with or without GM-CSF as an adjuvant cytokine. Ten patients with a significant proliferative T cell response against rCEA were selected for T cell epitope mapping. Fifteen-aa-long overlapping peptides covering the entire aa sequence of the external domain of CEA were used in a proliferation assay. In six of the patients a repeatable T cell response against at least one peptide was demonstrated. For the first time, nine functional HLA-DR epitopes of CEA were defined. Two of the peptides were recognized by more than one patient, i.e., two and three patients, respectively. Those 15-mer peptides that induced a proliferative T cell response fitted to the actual HLA-DR type (SYFPEITHI). The affinity of the native peptides for the T cell receptor was in the low to intermediate range (scores 6–19). The 15-mer peptides also contained 9-mer peptide sequences that could be predicted to bind to the actual HLA-ABC genotypes (SYFPEITHI/BIMAS). Blocking experiments using monoclonal antibodies indicated that the proliferative T cell response was both MHC class I and II restricted. The defined HLA-DR T cell epitopes were spread over the entire CEA molecule, but a higher frequency was noted towards the C-terminal. Peptides with a dual specificity may form a basis for production of subunit cancer vaccines, but modifications should be done to increase the T cell affinity, thereby optimizing the antitumoral effects of the vaccine. [ABSTRACT FROM AUTHOR]

T-cell immune dysfunction in patients with malignant tumours has been attributed to the altered expression of components of the T-cell receptor (TCR)/CD3 complex and their associated intracellular protein tyrosine kinases. In this study, four-colour flow cytometry was applied to study the surface bound molecules TCR α β, CD28, CD152 and CD154 involved in T-cell signalling and the signal transduction molecules CD3 ζ, p56lck, p59fyn, ZAP-70 and phosphatidyl-inositol-3 kinase (PI3-k) as well as the intracellular cytokines interferon- γ (IFN- γ), interleukin (IL)-4 and IL-2 as a functional read-out of non-stimulated and superantigen (staphylococcus enterotoxin B)-stimulated blood T cells of multiple myeloma (MM) patients at different stages of the disease. Multiple abnormalities were demonstrated in the CD4 and CD8 populations, both under non-stimulated and superantigen-stimulated conditions. There was a marked reduction, particular in advanced stage MM, in the proportion of CD4 and CD8 cells expressing CD28, CD152, CD3 ζ, p56lck, ZAP-70 and PI3-k. The level of intracellular T-cell cytokines (IFN- γ, IL-2 and IL-4) was normal or increased in non-stimulated cells but activation-induced cytokine production was impaired. These results illustrated profound and multiple T-cell signalling defects, from the surface and down-stream, consistent with involvement of a master T-cell function, especially in advanced stage MM. These data should be taken into consideration when developing immune-based therapeutic approaches and when applying new emerging technologies that aim to restore T-cell functions. [ABSTRACT FROM AUTHOR]

Background: Hemorrhagic cystitis (HC) in allogeneic bone marrow transplanted (BMT) patients is associated with BK virus (BKV) reactivation manifested as BK viruria. However, since 77–90% of all adult BMT patients excrete BKV, viral reactivation alone cannot be responsible for HC. Recently, a significant overrepresentation of C→G mutations in the Sp1 binding site in the non-coding control region (NCCR) of BKV was shown to be present in HC patients and absent in non-HC patients. Objectives: We aimed to investigate if this mutation resulted in excessive BKV excretion in HC patients. Study design: A Real-Time PCR was developed and used to quantify BKV in urine samples from 21 patients with HC, with and without the mutations, as well as from patients without HC. Results: Quantification of BKV was successful in 18 of 21 urine patients (six with and six without C→G mutations) and six patients without HC. A mean of 3.0×106 BKV copies/μl was detected in urine samples of HC patients with C→G mutations, compared to a mean of 1.5×106 BKV copies/μl in HC patients without C→G mutations and a mean of 1.0×106 BKV copies/μl in patients without HC. The obtained differences were however not statistically significant, due to one individual non-HC patient with an extremely high BKV copy number. Nevertheless, while 50% of the samples in the HC groups expressed 1×106 copies/μl or more, only one of the samples in the non-HC group contained a virus quantity higher than 5×105 copies. Conclusions: Although we could not confirm that the C→G mutations in the Sp1 site of BKV were responsible for an increased viral load in patients with HC, our data suggest that levels of BKV above 104 copies/μl may indicate a risk for HC. [Copyright &y& Elsevier]

Abstract: Analysis of cytokine production is a tool to functionally characterise T cells. In this study, spontaneous and polyclonal activation induced cytokine production in T cells were assessed by flow cytometry in patients with B-CLL. Patients with progressive disease had a significantly increased number of T cells spontaneously producing IL-2, IL-4 and GM-CSF as compared to healthy donors and patients with non-progressive CLL, which was not the case for TNF-α and IFN-γ producing T cells. However, no difference in the frequency of T cells producing these cytokines was seen comparing patients with non-progressive disease to control donors. Polyclonal activation of B-CLL T cells in vitro induced an increased proportion of T cells producing these five cytokines in patients as well as in control donors, indicating that T cells in CLL patients might have a relatively well preserved functional capacity. However, the increase in GM-CSF, TNF-α and IL-4 producing T cells was more marked in CLL patients than in controls. Furthermore, following activation, a higher frequency of cytokine-producing T cells was noted in patients with progressive disease as compared to those with non-progressive disease. The augmented number of cytokine-producing T cells in CLL may indicate an up-regulated capability of T cells to secrete cytokines, especially in patients with progressive CLL. The increased production of the T cell derived cytokines GM-CSF, TNF-α, IL-4 and IL-2 is interesting, as these cytokines have previously been shown to support growth of B-CLL leukaemic cells in vitro and as T cells might specifically recognise the autologous leukaemic B cells in vivo . The findings may suggest a role for T cells in the pathogenesis of B-CLL. [ABSTRACT FROM AUTHOR]

Objectives: Antigen expression intensity is becoming important for decision-making in relation to monoclonal antibody therapy. By quantifying CD20, CD22 and CD52 expression on chronic lymphocytic leukemia and normal (control) B cells, over time. The effect of Interleukin-4 therapy on CD20 antigen intensity on B-CLL cells in vivo was also determined. Methods: Lymphocytes were purified at weeks 0, 4 and 8 from five B-CLL patients, five healthy volunteers and seven B-CLL patients receiving IL-4 therapy. The number of antigen receptor sites was calculated in molecules of equivalent soluble fluorochrome using flow cytometry. Results: The mean number of CD20 receptors at baseline was significantly lower on B-CLL cells compared to normal B cells (8160 vs 87 046; P < 0.0001). Similar results were obtained for CD22 (8630 vs 27 647; P < 0.01), but not for CD52 (371 303 vs 409 484; P=0.54). When soluble fluorochrome values at weeks 4 and 8 were analysed as change in per cent from baseline (Δ%), there was < 10 Δ% variability in CD20 expression on control B cells, but considerable variability (22.5 - 67.5 Δ%) on B-CLL cells. Expression of CD22 in CLL and control B cells varied by < 15 Δ%. CD52 on CLL B cells showed slightly greater variability (±35 Δ%) than that of CD22 (± 15Δ%), but less than that of CD20. IL-4 therapy did not consistently increase the CD20 expression on B-CLL cells in vivo. Conclusion: Our data confirm differences in intensity between different target antigens on B-CLL cells, and draws attention to the fact that a substantial variability may occur over time, which may influence clinical decision-making. Caution must be taken when interpreting in vitro results on cytokine-mediated receptor intensity up-regulation. [ABSTRACT FROM AUTHOR]

Abstract: Clinical progression of B-cell chronic lymphocytic leukemia (B-CLL) depends on survival and accumulation of leukemic cells, regulated in part by physical cell contact and soluble molecules. Here we have studied the Fas/FasL system in relation to clinical progression in B-CLL. Serum levels of soluble Fas (sFas) and FasL (sFasL) were determined by ELISA in 43 progressive and 40 non-progressive B-CLL patients and in 21 control individuals. Correlation between sFas serum levels and clinical progression, stage and survival were statistically analyzed. We found high levels of sFas in B-CLL sera correlated with disease progression (p<0.01). In addition, higher sFas levels were found in patients in stages II, III and IV in comparison to patients in stage 0 (p<0.05, p<0.01, p<0.03, respectively). Survival was significantly shorter for patients with >=6 ng/ml sFas serum levels, although a multivariate analysis did not show sFas to be a significant independent prognostic factor. Fresh B-CLL cells showed only low levels of membrane expression, which were not correlated to sFas levels in serum. In vitro activation of B-CLL cells increased Fas expression, as reported earlier, and induced cells to release sFas into the supernatant. In conclusion, our results indicate that sFas in serum may be a useful parameter for the prediction of clinical progression in B-CLL. [ABSTRACT FROM AUTHOR]

This study analysed a naturally occurring specific cellular immunity against tumour cells in chronic lymphocytic leukaemia (CLL) patients. Five out of eight patients had blood T lymphocytes able to recognize spontaneously and specifically the autologous tumour B cells (proliferation assay). In these five patients, detection of cytokines by real-time reverse transcription polymerase chain reaction (RT-PCR) revealed that granulocyte–macrophage colony-stimulating factor (GM-CSF) was the most abundant cytokine gene expressed by the T cells that recognized the autologous tumour B cells. Other activated cytokine genes were γ-interferon (IFN), interleukin (IL)-2 and tumour necrosis factor (TNF)-α, but not IL-4. This profile suggests a type 1 anti-B-CLL T-cell response. CD80 and CD54 were relatively downregulated on the native tumour B cells compared with control normal B cells. Upregulation of CD80 on the leukaemic cells was mandatory for the induction of such a specific T-cell response. CD80 and CD54 monoclonal antibodies inhibited the specific T-cell DNA synthesis proliferation. The proliferative T-cell response was either MHC class I or class II restricted (inhibition by monoclonal antibodies). The specific cytokine gene expression could be found in isolated CD4, as well as CD8, T-cell subsets. This study demonstrated the presence of a potential natural specific CD4, as well as a CD8 type 1 T-cell immunity against the leukaemic CLL tumour B cells in CLL. A further detailed analysis of the spontaneous anti-CLL T-cell immunity is warranted that may facilitate the development of effective anti-tumour vaccines in CLL. [ABSTRACT FROM AUTHOR]

We have previously shown that autologous T cells recognize leukaemic cells from patients with chronic lymphocytic leukaemia (B-CLL) in an MHC class I- and/or II-restricted manner. A candidate recognition structure might be the tumour cell-derived Ig VH complementarity-determining region (CDR)3. Three patients with B-CLL were analysed for the presence of autologous T cells recognizing the tumour-specific VH-CDR3 region. The VH region was shown to be mutated in all three patients. In two patients, a VH-CDR3-specific T-cell response was detected by proliferation assay, as well as by γ-interferon (IFN) production. The responses could be inhibited by monoclonal antibodies against MHC class II, but not MHC class I. In the third patient, a VH-CDR3 proliferative response was detected, which could be inhibited by an anti-MHC class I monoclonal antibody, but not by anti-MHC class II antibodies. No γ-IFN response could be detected in this patient. In no patient was an interleukin (IL)-4 response noted. Thus, in patients with B-CLL, naturally occurring T cells recognizing the tumour-unique VH-CDR3 region are present. [ABSTRACT FROM AUTHOR]

Phosphorylation of receptor tyrosine kinases (RTKs) has a key role in cellular functions contributing to the malignant phenotype of tumor cells. We and others have previously demonstrated that RTK ROR1 is overexpressed in chronic lymphocytic leukemia (CLL). Silencing siRNA downregulated ROR1 and induced apoptosis of CLL cells. In the present study we analysed ROR1 isoforms and the phosphorylation pattern in CLL cells (n=38) applying western blot and flow-cytometry using anti-ROR1 antibodies and an anti-phospho-ROR1 antibody against the TK domain. Two major ROR1 bands with the size of 105 and 130 kDa respectively were identified, presumably representing unglycosylated (immature) and glycosylated (mature) ROR1 respectively as well as a 260 kDa band which may represent dimerized ROR1. A ROR1 band of 64 kDa that may correspond to a C-terminal fragment was also noted, present only in the nucleus. The 105 kDa ROR1 isoform was more frequently expressed in non-progressive as compared to progressive CLL patients (p=0.03). The 64, 105, 130 and 260 kDa bands were constitutively phosphorylated both at tyrosine and serine residues. Phosphorylation intensity of the mature (130 kDa) isoform was significantly higher in progressive than in non-progressive disease (p<0.001). Incubation of CLL cells with a mouse anti-ROR1 KNG or an anti-ROR1 CRD mAb respectively induced dephosphorylation of ROR1 before entering apoptosis. In conclusion CLL cells expressed different isoforms of ROR1 which were constitutively phosphorylated. The mature, phosphorylated ROR1 isoform was associated with a progressive disease stage. Targeting ROR1 by mAbs induced specific dephosphorylation and leukemic cell death. ROR1 might be an interesting therapeutic target. [ABSTRACT FROM AUTHOR]