Clinical and Immune Effects of Lenalidomide in Combination with Gemcitabine in Patients with Advanced Pancreatic Cancer.

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 NCT01547260.

In vitro immunomodulatory properties of gemcitabine alone and in combination with interferon-alpha.

In general, conventional chemotherapy is associated with significant toxicity leading to immunosuppression manifesting mainly in the lymphocyte depletion. This immunosuppression promotes tumor growth and elicits the tumor cell dissemination. However, chemotherapy can be immune stimulative especially in combination with an immunotherapy. In this work, we investigated in vitro effects of gemcitabine alone and in combination with interferon-alpha on splenocytes obtained from healthy and pancreatic carcinoma bearing mice. We showed that gemcitabine alone depletes the regulatory T cells in the splenocyte culture. Gemcitabine in combination with interferon-alpha demonstrated some immunomodulatory features, but these effects were interferon-alpha dependent. We concluded that combination of both drugs induces rather cumulative effects, supposing that these therapeutic could be applied together for a chemo-immunotherapy.

Generation of AcGFP fusion with single-chain Fv selected from a phage display library constructed from mice hyperimmunized against 5-methyl 2'-deoxycytidine.

DNA methylation is involved in many diseases such as cancer and autoimmunity. We generated recombinant single-chain Fv (scFv) antibodies against 5-methyl-2'-deoxycytidine (m(5)dCyd) using phage display technology and a hyperimmunized mouse, and the scFv of most interest were constructed as fusion proteins with green fluorescent protein obtained from Aequorea coerulescens GFP (AcGFP). Using RNA isolated from mouse spleens, we constructed a scFv library consisting of λ light chains. The scFv library was selected against m(5)Cyd-BSA and enriched through four rounds of panning. The scFv library was concentrated about 390-fold and an individual clone was reacted with m(5)Cyd-BSA. Two scFvs with high reactivity for m(5)Cyd-BSA termed 1-2 and 1-12 were produced. Furthermore, methylated DNA-binding activities of the scFvs were confirmed using an indirect immunofluorescence assay. Additionally, N- and C-terminal scFv 1-2 fusion with AcGFP were constructed, and we observed the N-terminal AcGFP exhibited much higher fluorescence intensity than the C-terminal fusions. The AcGFP-scFv 1-2 modified N-terminus of scFv with AcGFP had high fluorescence intensity, but the scFv 1-2-AcGFP modified C-terminus of scFv with AcGFP had low fluorescence intensity. The cross-reactivity of AcGFP-scFv 1-2 was similar to scFv 1-2, and thus, AcGFP-scFv 1-2 could be used in a direct immunofluorescence assay. The scFv fusion proteins may be useful for the detection and quantification of cellular methylated DNA in various specimens.

Generation of a recombinant single-chain variable fragment (scFv) targeting 5-methyl-2'-deoxycytidine.

We generated a single-chain variable fragment (scFv) against 5-methyl-2'-deoxycytidine (m(5)dCyd) using phage display technology. The heavy and light chain variable region genes were amplified by the polymerase chain reaction (PCR) from hybridoma cell line FMC9 and assembled as an scFv fragment with a flexible linker (Gly(4)-Ser)(3). The scFv DNA fragment was then cloned into pCANTAB-5E, and a phage displaying the scFv was produced. Antigen-positive phage clones were successfully selected by enzyme-linked immunosorbent assay (ELISA). The scFv was modified with FLAG and His tags for detection and purification. The scFv reacted strongly with m(5)dCyd and weakly with 5-methylcytidine (m(5)Cyd) but not with cytidine (Cyd) and 1-methyladenosine in a manner similar to the monoclonal antibody (MoAb). Although the specificities of scFv and MoAb were almost identical, the sensitivity of the scFv (IC(50) 0.054 microg/ml) was approximately 80 times higher than that of the parent MoAb (IC(50) 4.27 microg/ml), determined by inhibition ELISA. As a biochemical application of this scFv, we quantified the m(5)dCyd content of genomic DNA by enzymatic hydrolysis using inhibition ELISA. The cancer cell lines HeLa, HeLa S3 and MDA-MB-453 contained approximately 1% of the methylated DNA in total genomic DNA, as did peripheral blood cell genomic DNA from healthy volunteers, but HT29 and T-47D showed hypomethylation compared with the HeLa, HeLa S3 and MDA-MB-453 cell lines. The scFv generated here may be applicable to the assessment of cellular DNA methylation levels and is more sensitive than the MoAb.

A highly sensitive enzyme immunoassay for evaluation of 2'-deoxycytidine plasma level as a prognostic marker for breast cancer chemotherapy.

A highly sensitive competitive enzyme immunoassay (EIA) has been developed and validated for the determination of the plasma level of 2'-deoxycytidine (dCyd), the potential prognostic marker for breast cancer chemotherapy. This assay employed a monoclonal antibody that recognizes dCyd with a high specificity, and 5'-succinyl-dCyd (5'sdCyd) conjugate of bovine serum albumin (5'sdCyd-BSA) immobilized onto microplate wells as a solid phase. The assay involved a competitive binding reaction between dCyd, in plasma sample, and the immobilized 5'sdCyd-BSA for the binding sites of the anti-dCyd antibody. The bound antibody was quantified with horseradish peroxidase-labeled anti-immunoglobulin second antibody and 3,3',5,5'-tetramethylbenzidine as a peroxidase substrate. The concentration of dCyd in the sample was quantified by its ability to inhibit the binding of the antibody to the immobilized 5'sdCyd-BSA and subsequently the color formation in the assay. The assay limit of detection was 8 nM and the effective working range at relative standard deviations (R.S.D.s) of

Novel monoclonal antibody recognition of oxidative DNA damage adduct, deoxycytidine-glyoxal.

Glyoxal, a reactive aldehyde, is a decomposition product of lipid hydroperoxides, oxidative deoxyribose breakdown, or autoxidation of sugars, such as glucose. It readily forms DNA adducts, generating potential carcinogens such as glyoxalated deoxycytidine (gdC). A major drawback in assessing gdC formation in cellular DNA has been methodologic sensitivity. We have developed an mAb that specifically recognizes gdC. Balb/c mice were immunized with DNA, oxidatively modified by UVC/hydrogen peroxide in the presence of endogenous metal ions. Although UVC is not normally considered an oxidizing agent, a UVC/hydrogen peroxide combination may lead to glyoxalated bases arising from hydroxyl radical damage to deoxyribose. This damaging system was used to induce numerous oxidative lesions including glyoxal DNA modifications, from which resulted a number of clones. Clone F3/9/H2/G5 showed increased reactivity toward glyoxal-modified DNA greater than that of the immunizing antigen. ELISA unequivocally showed Ab recognition toward gdC, which was confirmed by gas chromatography-mass spectrometry of the derivatized adduct after formic acid hydrolysis to the modified base. Binding of Ab F3/9 with glyoxalated and untreated oligomers containing deoxycytidine, deoxyguanosine, thymidine, and deoxyadenosine assessed by ELISA produced significant recognition (p > 0.0001) of glyoxal-modified deoxycytidine greater than that of untreated oligomer. Additionally, inhibition ELISA studies using the glyoxalated and native deoxycytidine oligomer showed increased recognition for gdC with more than a 5-fold difference in IC(50) values. DNA modified with increasing levels of iron (II)/EDTA produced a dose-dependent increase in Ab F3/9 binding. This was reduced in the presence of catalase or aminoguanidine. We have validated the potential of gdC as a marker of oxidative DNA damage and showed negligible cross-reactivity with 8-oxo-2'-deoxyguanosine or malondialdehyde-modified DNA as well as its utility in immunocytochemistry. Formation of the gdC adduct may involve intermediate structures; however, our results strongly suggest Ab F3/9 has major specificity for the predominant product, 5-hydroxyacetyl-dC.

Fluorouracil for allergic reactions to capecitabine.

OBJECTIVE: To report the safe use of fluorouracil in a patient with breast cancer who had allergic reactions to capecitabine. CASE SUMMARY: A 42-year-old African American woman with metastatic breast cancer developed progressive disease. Capecitabine 1500 mg taken by mouth twice daily was prescribed as the salvage chemotherapy. She developed a generalized rash and itching, sore throat, and dizziness approximately 4 hours after the first dose of capecitabine. These reactions recurred immediately after the second dose. Capecitabine was discontinued and the allergic reactions resolved after the woman took diphenhydramine for 1 week. In view of limited therapeutic options for her progressive disease, a trial of fluorouracil 300 mg/m(2)/d continuous intravenous infusion over 5 days was initiated without any premedications. She did not experience any reactions. The dose of fluorouracil in the second cycle was increased to 400 mg/m(2)/d continuous infusion over 5 days. DISCUSSION: Capecitabine is not intrinsically cytotoxic, but is converted to fluorouracil in tumor tissues via a 3-step enzymatic pathway. Capecitabine reaches peak blood concentrations in about 1.5 hours, with peak fluorouracil concentrations occurring at 2 hours. The elimination half-life of both drugs is 0.5-0.7 hours. The patient tolerated the rechallenge with fluorouracil without complications. Objective causality assessment revealed that the adverse event was probably drug induced. It was postulated that the allergic reaction was most likely caused by capecitabine or the intermediate metabolites based on the immediate reappearance of symptoms from the rechallenge, pharmacokinetic data, and well-tolerance of fluorouracil. CONCLUSIONS: The use of fluorouracil treatment with careful monitoring can be considered in a patient with mild allergic reactions to capecitabine.

Preparation of a monoclonal antibody specific for 5-methyl-2'-deoxycytidine and its application for the detection of DNA methylation levels in human peripheral blood cells.

A monoclonal antibody specific for a modified nucleoside, 5-methyl-2'-deoxycytidine (m5dCyd), was prepared using 5-methylcytidine (m5Cyd)-keyhole limpet haemocyanin (KLH) conjugate, and was characterized. Termed FMC9, the antibody reacts with m5dCyd and slightly with m5Cyd and 5-methylcytosine (m5Cyt) but not with other nucleosides tested in this investigation. FMC-9 was used in an enzyme-linked immunosorbent assay (ELISA) system for the quantitation of m5dCyd levels. Sensitivity was in the picomole range. Methylation levels in peripheral blood cells of healthy donors were determined by inhibition ELISA. The percentage of m5dCyd in peripheral blood cells of 10 healthy donors was 5.08 +/- 0.50%. These results suggest that the inhibition ELISA using FMC9 is useful to monitor m5dCyd levels in the peripheral blood cells.

Monoclonal antibody affinities of structurally related modified nucleosides.

A scale of relative affinities of a series of 2'-deoxycytidine and cytidine (CD) derivatives was established based on the data of cross-reactivities of these compounds as well as the displacements obtained from a competitive ELISA. No correlation could be established between the nucleosides modifying structures and the affinities. This can be explained by the possibilities of the modifying structures of intra- and intermolecular nonimmunospecific interactions owing to their degree of functionalization.

Immunochemical detection of urinary 5-methyl-2'-deoxycytidine as a potential biologic marker for leukemia.

A monoclonal antibody against 5-methylcytidine was prepared and characterized. This antibody, termed AMC, was reactive with compounds that had 5-methylcytosine structure (i.e. 5-methyl-2'-deoxycytidine, 5-methylcytidine and 5-methylcytosine). AMC had the highest reactivity to 5-methyl-2'-deoxycytidine among reactive compounds and had no or very slight cross-reactivity to cytidine-related compounds and any other compounds. Analysis of immunoreactive materials in urine revealed that 5-methyl-2'-deoxycytidine rather than 5-methylcytidine was, contrary to our expectation, the major component. Then the inhibition ELISA system using AMC was established and urinary levels of 5-methyl-2'-deoxycytidine in healthy individuals and cancer patients were determined. The mean excretion levels of healthy individuals was 0.90 +/- 0.43 nmol/mumol creatinine and the cut-off value was set at the mean + 2 S.D. of healthy individuals (1.76 nmol/mumol creatinine). Among various types of cancer tested, elevated levels of 5-methyl-2'-deoxycytidine were detected in leukemia patients. From these results, urinary 5-methyl-2'-deoxycytidine might be applicable as a biologic marker for leukemia.

Time-resolved fluoroimmunoassay of 5-methyl-2'-deoxycytidine employing europium-labeled antigen as tracer.

We describe a solid-phase fluoroimmunoassay, based on competition between europium-labeled 5 MeCyd (5-methylcytidine) and sample 5MedCyd (5-methyl-2'-deoxycytidine) for polyclonal anti-5MedCyd antibodies (rabbit). Europium labeling of antigen was performed using a novel polylysine-5MeCyd conjugate. Standard and sample preparations containing 5MedCyd inhibited the binding of the europium-labeled 5MeCyd to the antibody molecules. A second antibody, directed against rabbit IgG, was coated on the solid phase, and bound the IgG-5MeCyd-polylysine-europium complex, giving rapid and complete separation of antibody-bound and free antigen. The measuring range was from 3.7 to 2500 pmol of 5MedCyd per assay. A good correlation between the results obtained with TR-FIA and HPLC was demonstrated when the methods were applied to the measurement of methylation in various DNA samples, enzymatically hydrolyzed to their constituent deoxyribonucleosides. This new TR-FIA possesses the same advantages (high sensitivity, wide assay range, rapidity, simplicity, and low cost) as the previous assay developed in our laboratories. The superiority of the new system is based on (i) its low inter- and intra-assay variation, (ii) low antiserum consumption, and (iii) a protocol, which permits the use of second-antibody-coated microtitration strips common to other assays.

[Selective inhibition of the primary humoral immune response in mice with a combination of deoxycytidine with Cytosar]. / Izbiratel'noe ugnetenie pervichnogo gumoral'nogo immunnogo otveta u myshei kombinatsiei dezoksitsitidina s tsitozarom.

Cytosine arabinoside administration in lethal doses to C57BL/6j female mice immunized with red blood cells leads, under deoxycytidine protection, to reduction of serum hemagglutinin level on day 5 without toxicosis. Simultaneous injection of the metabolite and the antimetabolite proves to be optimum.