Reactions of 4-[Bis(2-chloroethyl)amino]benzenebutanoic acid (chlorambucil) with DNA.

4-[Bis(2-chloroethyl)amino]benzenebutanoic acid (=chlorambucil, 1; 2.5 mM) was allowed to react with single- and double-stranded calf thymus DNA at physiological pH (cacodylic acid, 50% base) at 37 degrees . The DNA-chlorambucil adducts were identified by analyzing the DNA hydrolysates by NMR, UV, HPLC, LC/ESI-MS/MS techniques as well as by spiking with authentic materials. ssDNA was more reactive than dsDNA, and the order of reactivity in ssDNA was Ade-N1>Gua-N7>Cyt-N3>Ade-N3. The most reactive site in dsDNA was Ade-N3. The Gua-N7 and Ade-N3 adducts were hydrolytically labile. Ade-N7 adduct could not be identified in the hydrolysates of ssDNA or dsDNA. The adduct Gua-N7,N7, which consists of two units of Gua bound together with a unit derived from chlorambucil, is a cross-linking adduct, and it was detected in the hydrolysates of ssDNA and dsDNA. Also several other adducts were detected which could be characterized by spiking with previously isolated authentic adducts or tentatively by MS. The role of chlorambucil-DNA adducts on the cytotoxicity and mutagenity of 1 is also discussed.

A New Generation Prothrombin Time Method for INR.

Prothrombin time (PT) is the leading test for monitoring oral anticoagulation therapy (OAT). We sought to determine INR taking into account only active coagulation factors FII, FVII and FX without inhibition in patient plasmas and calibrator kits.We measured PT using a combined thromboplastin reagent. The calculation was based on a new PT method, which measures active coagulation factors (F II, F VII, FX) and corrects the errors caused by inactive coagulation factors.On this basis, an INR result with and without inhibition for individual patient samples was also calculated and applied to 200 plasma samples obtained from OAT patients. Conspicuous variation in inhibition between the four calibration kits was noted. The kinetics of this inhibition was closest to a noncompetitive pattern.The need of correction for INRs of single patients increases with higher INRs. At the same level of patient INRs the coagulation inhibiton varies markedly.It has been known that different thromboplastin reagents possess variable sensitivities, but this may depend on sensitivity in inactive coagulation factors. PT methods today measure the sum of active coagulation factors and inhibition of inactive coagulation factors. ISI calibrators contain variable amounts of inactive coagulation factors, which renders harmonisation of INR results.Application of the Acf-PT (INR(Acf)) presented in this work develops the PT methodology to measure the true coagulation activity in vivo for patient warfarin therapy without inhibition. INR(Inh) can evidently also be used for the diagnostics and follow-up of certain liver diseases.

Antibody response to 7-valent conjugated pneumococcal vaccine in patients with chronic lymphocytic leukaemia.

Chronic lymphocytic leukaemia (CLL) is a common adulthood mature B-cell neoplasm. Infections are the most important cause of mortality in this condition, and Streptococcus pneumoniae has been considered the most important single pathogen. We investigated the immunogenicity of 7-valent pneumococcal conjugate vaccine in patients with CLL. The study material comprised 52 patients with CLL and 25 age- and sex-matched controls. The subjects were vaccinated with Prevenar pneumococcal conjugate vaccine. Serum samples were taken for antibody determinations before and four weeks after vaccination. Antibody response rates to vaccine antigens were lower in patients with CLL compared to controls. However, if the vaccine had been administered at an early stage of the disease, i.e. before commencement of chemotherapy and the development of hypogammaglobulinaemia, a significant vaccination response to at least six antigens was obtained in almost 40% of the CLL patients. Our results indicate that early administration of conjugate vaccine may be beneficial in CLL.

Reactions of {4-[bis(2-chloroethyl)amino]phenyl}acetic acid (phenylacetic acid mustard) with 2'-deoxyribonucleosides.

Phenylacetic acid mustard (PAM; 2), a major metabolite of the anticancer agent chlorambucil (CLB; 1), was allowed to react with 2'-deoxyadenosine (dA), 2'-deoxyguanosine (dG), 2'-deoxycytidine (dC), 2'-deoxy-5-methylcytidine (dMeC), and thymidine (T) at physiological pH (cacodylic acid, 50% base). The reactions were followed by HPLC and analyzed by HPLC/MS and/or (1)H-NMR techniques. Although the predominant reaction observed was hydrolysis of PAM, 2 also reacted with various heteroatoms of the nucleosides to give a series of products: compounds 5-31. PAM (2) was found to be hydrolytically slightly more stable than CLB (1). The principal reaction sites of 2 with dA, dG, and with all pyrimidine nucleosides were N(1), N(7), and N(3), resp. Also, several other adducts were detected and characterized. There was no significant difference in the reactivity of 1 and 2 with dG, dA or T, but the N(3) dC-PAM adduct was deaminated easier than the corresponding CLB derivative. The role of PAM-2'-deoxyribonucleoside adducts on the cytotoxic and mutagenic properties of CLB (1) is discussed.

Two novel nucleoside ester derivatives of chlorambucil as potential antileukemic prodrugs: a preliminary study.

2-Chloro-2'-deoxyadenosine (cladribine) and chlorambucil are two drugs used in the treatment of lymphoid malignancies. We have synthesized 5'-O-esters of cladribine and its parental nucleoside 2'-deoxyadenosine with chlorambucil (2-chloro-2'-deoxyadenosine-chlorambucil and 2'-deoxyadenosine-chlorambucil, respectively) and compared some properties of the esters with regard to their potential use as antileukemic prodrugs. The 5'-O-ester bond showed no spontaneous hydrolysis at pH 7.4, but was susceptible to hydrolysis by porcine liver esterase and enzymes present in human lymphocyte lysate and blood plasma. Both 2-chloro-2'-deoxyadenosine-chlorambucil and 2'-deoxyadenosine-chlorambucil were taken up more avidly than their parental nucleosides by normal and malignant human lymphoid cells. 2-Chloro-2'-deoxyadenosine-chlorambucil was by an order of magnitude more toxic than 2'-deoxyadenosine-chlorambucil to human leukemic MOLT4 cells in culture. On the other hand, 2-chloro-2'-deoxyadenosine-chlorambucil cytotoxicity did not exceed that of its parental 2-chloro-2'-deoxyadenosine in MOLT4 cells, whereas 2'-deoxyadenosine-chlorambucil was considerably more cytotoxic than free chlorambucil in a variety of myeloid and lymphoid human malignant cell lines. Moreover, acute toxicity of 2'-deoxyadenosine-chlorambucil was lower than that of chlorambucil in mice. In summary, 2'-deoxyadenosine-chlorambucil, but not 2-chloro-2'-deoxyadenosine-chlorambucil, shows promise for clinical utility as a chlorambucil prodrug and thus warrants a more detailed study in vivo.

Strikingly homologous immunoglobulin gene rearrangements and poor outcome in VH3-21-using chronic lymphocytic leukemia patients independent of geographic origin and mutational status.

We recently reported that Swedish VH3-21-using chronic lymphocytic leukemia (CLL) patients showed restricted immunoglobulin gene features and poor prognosis despite VH mutation status. To investigate this further, we analyzed the VH and VL gene rearrangements in 90 VH3-21+ patients from Sweden, Germany, Italy, United States, Finland, and Australia and correlated these data with survival and other prognostic markers. Sixty-three percent exhibited mutated VH genes and 37% unmutated VH genes. Fifty (56%) patients displayed a short and homologous heavy-chain CDR3, many of these with the amino acid motif DANGMDV. Also, a highly biased Vlambda2-14 use was evident in 72% of patients with a restricted light-chain CDR3, QVWDS(S/G)SDHPWV. Combined restricted heavy- and light-chain CDR3s were found in patients from all included countries. Although VH3-21+ CLLs have a remarkably predominant lambda expression, analyses of kappa deleting element indicated a conserved light-chain rearrangement order. The overall survival was poor in the VH3-21+ cohort (median survival, 88 months), with no significant difference in relation to mutation status or CDR3 homology. High ZAP-70 and CD38 expression was found in both mutated and unmutated VH3-21+ cases as well as a slight increase of 11q-aberrations. In summary, highly restricted B-cell receptors and worse outcome characterize VH3-21+ CLLs independent of geographic origin and mutation status.

Mitogen induced activation, proliferation and surface antigen expression patterns in unmutated and hypermutated chronic lymphocytic leukemia cells.

OBJECTIVES: To determine whether the immunoglobulin V(H) gene mutational status has an effect on the activation, proliferation and surface antigen expression of chronic lymphocytic leukemia (CLL) cells when stimulated in vitro. METHODS: The proliferation and activation responses of CLL cells were studied in 22-immunoglobulin gene V(H) unmutated (UM-CLL) and 12 hypermutated (M-CLL) CLL cases in 4-day cultures. As the mitogen responses have been previously shown to be diverse in CLL, a case-specific strategy based on optimized mitogen combinations (OMCs) of interleukin-2 (IL-2), 12-O-tetradecanoylphorbol 13-acetate (TPA), Staphylococcus aureus Cowan 1 (SAC), and human recombinant tumor necrosis factor alpha (TNF) was applied in cell stimulation. The expression of 23 surface membrane antigens (CD5, CD11c, CD19, CD20, CD21, CD22, CD23, CD25, CD27, CD38, CD40, CD45, CD45RA, CD45RO, CD79b, CD80, CD95, CD124, CD126, CD130, FMC7, IgD, and IgM) was studied by flow cytometry at days 0 and 4. RESULTS: The proliferation and activation responses were similar in UM-CLL and M-CLL when OMCs contained IL-2, TPA or TNF. SAC induced faster proliferation in UM-CLL than in M-CLL. OMC stimulation induced preferential down-regulation of growth- promoting cell surface receptors CD5, CD21, and CD124 and preferential up-regulation of growth-inhibiting antigen CD80 in M-CLL. CONCLUSIONS: Difference in immunophenotypic evolution of UM-CLL and M-CLL can be demonstrated if appropriate matrix signals are provided. The pathways for CD5, CD21, CD124 (IL4R), and CD80 (B7-1) regulation should be further explored in relation with somatic hypermutation and outcome of CLL.

Patients with chronic lymphocytic leukemia with mutated VH genes presenting with Binet stage B or C form a subgroup with a poor outcome.

BACKGROUND AND OBJECTIVES: The immunoglobulin VH gene mutation status is a strong prognostic indicator in B-cell chronic lymphocytic leukemia (CLL), since unmutated VH genes are correlated with short survival. However, the traditional cut-off level dividing mutated and unmutated cases, i.e. more or less than 2% mutations, has been questioned and other cut-offs have been suggested. We investigated whether an alternative cut-off should be applied and the relation of mutational status to another prognostic marker, Binet staging. DESIGN AND METHODS: VH gene mutation status was assessed in 332 CLL cases by polymerase chain reaction amplification and nucleotide sequencing and was further correlated with overall survival using different VH mutation cut-offs (1-7%) and Binet stage. RESULTS: After testing different mutation borders, the 2% cut-off remained the best discriminative level for determining prognosis. Interestingly, prognostic stratification was improved by combining the information on VH gene mutation status with that of Binet stage: unmutated cases (all stages, n=151, mutated cases with stage A (n=77), and mutated cases with stage B or C (n=37) had a median survival of 82, 179 and 74 months, respectively. INTERPRETATION AND CONCLUSIONS: CLL cases displaying mutated VH genes with Binet stage B or C had a survival similar to that of unmutated cases and significantly shorter than that of mutated stage A CLL. Our result reveals clinical heterogeneity within the VH mutated CLL group by inclusion of Binet stage data, a finding which is of importance when considering surrogate marker(s) for VH mutation status.

Poor agreement among prothrombin time international normalized ratio methods: comparison of seven commercial reagents.

BACKGROUND: Prothrombin time (PT) has long been the most popular test for monitoring oral anticoagulation therapy. The International Normalized Ratio (INR) was introduced to overcome the problem of marked variation in PT results among laboratories and the various recommendations for patient care. According to this principle, all reagents should be calibrated to give identical results and the same patient care globally. This is necessary for monitoring of single patients and for application of the results of anticoagulation trials and guidelines to clinical practice. METHODS: We took blood samples from 150 patients for whom oral anticoagulation had been prescribed. Plasmas were separated and PTs determined by use of seven commercial reagents and four calibrator sets. The differences in results were assessed by plotting, for each possible pair of methods, the differences in INR values for each sample against the mean INR value (Bland-Altman difference plots). RESULTS: Mean results differed significantly (P <0.001) for 17 of 21 possible paired comparisons of methods. Only two pairs of methods produced very similar results when assessed for problems of substantial differences in INR values; a significant, systematic increase in the difference with INR; and a significant systematic increase in the variation in difference with increasing INR values. CONCLUSIONS: The agreement among several (and perhaps most) commercial INR methods is poor. The failure of current calibration strategies may severely compromise both the monitoring of individual patients and the application of oral anticoagulation guidelines and trial results to clinical practice.

Similar humoral immunity parameters in chronic lymphocytic leukemia patients independent of VH gene mutation status.

Chronic lymphocytic leukemia (CLL) is a clonal B-cell disorder, which has recently been divided into 2 subtypes based on the somatic hypermutation status of the immunoglobulin heavy chain (IgVH) genes. In patients with unmutated tumor cells the survival time is approximately half of that in mutated cases, but the reason for this difference is poorly understood. Since infections are the major cause of mortality in CLL, we investigated the effect of the mutation status on host immunity and proneness to infections in patients with CLL. As expected, the disease progression seemed to be faster and the disease more advanced (Binet B and C) among unmutated patients than in the mutated ones. Surprisingly, no differences in humoral immunity [immunoglobulin G (IgG), IgM, IgA, IgG subclasses, anti-ABO blood group antibodies and mannan-binding lectin (MBL)] or immune responses (Haemophilus influenzae serotype b conjugate vaccination) were detected between these 2 patient groups. Furthermore, UM-patients were not more prone to infections compared to M-patients, and therapy had no impact on the incidence and pattern of infections in either of the patient groups. The current findings within this patient cohort reveal that the worse outcome in the unmutated subgroup is not caused by more severe defects in immunity and increased susceptibility to infections when compared with the hypermutated group. It is thus conceivable that active immunization procedures such as vaccination can successfully be applied on patients with unmutated IgVH gene and advanced disease stage.

Novel adamantylated pyrimidines and their preliminary biological evaluations.

The synthesis of adamantylated pyrimidines was based on the reaction of 3-(adamantan-1-yl)-3-oxopropionic acid ethyl ester with urea, thiourea, guanidine as well as acetamidine, respectively. Then the compounds obtained were converted into respective bromo-, thio- and S-alkyl derivatives. The molecular structures for some compounds were studied by X-ray methods. The significant anticancer and antimicrobial properties of [2-(6-adamantan-1-yl-2-methylpyrimidin-4-ylthio)ethyl]dimethylamine were found.

Different gene expression in immunoglobulin-mutated and immunoglobulin-unmutated forms of chronic lymphocytic leukemia.

The mutation status of the immunoglobulin heavy chain variable regions (IgVH) has been found to be a good prognostic indicator for B-cell chronic lymphocytic leukemia (CLL) because unmutated VH genes are associated with rapid disease progression and shorter survival time. To study the differences in gene expression between the Ig-unmutated and Ig-mutated CLL subtypes, we performed gene expression profiling on 31 CLL cases and investigated the VH gene mutation status by sequencing. The array data showed that the greatest variances between the unmutated (20 cases) and the mutated (11 cases) group were in expressions of ZAP70, RAF1, PAX5, TCF1, CD44, SF1, S100A12, NUP214, DAF, GLVR1, MKK6, AF4, CX3CR1, NAFTC1, and HEX. ZAP70 was significantly more expressed in the Ig-unmutated CLL group, whereas the expression of all the other genes was higher in the Ig-mutated cases. These results corroborate a recent finding, according to which the expression of ZAP70 can predict the VH mutation status and suggest that RAF1, PAX5, and other differentially expressed genes may offer good markers for differentiating unmutated cases from mutated cases and thus serve as prognostic markers.

Subsets with restricted immunoglobulin gene rearrangement features indicate a role for antigen selection in the development of chronic lymphocytic leukemia.

We recently identified a chronic lymphocytic leukemia (CLL) subgroup using the immunoglobulin variable heavy-chain (V(H)) gene V(H)3-21 with almost identical heavy-chain complementarity determining region 3s (HCDR3s) and preferential variable light-chain (V(L)) gene usage, suggesting recognition of a common antigen epitope in this subset. To further explore the B-cell receptors (BCRs) in CLL, we characterized 407 V(H) rearrangements amplified from 346 CLLs regarding V(H), diversity (D), and joining (J(H)) gene usage and performed multiple alignment of the HCDR3 sequences. These analyses revealed 3 small subsets (2 V(H)1-69 groups, 7 cases; and 1 V(H)1-2 group, 5 cases) with highly restricted HCDR3 features including identical V(H)/D/J(H) usage, HCDR3 lengths, and shared N-sequences, in addition to the V(H)3-21 group (22 cases). Furthermore, another 3 groups (9 V(H)1-3(+) cases, 3 V(H)1-18(+) cases, and 5 V(H)4-39(+) cases) had essentially identical V(H)/D/J(H) use and similar HCDR3 lengths but less conserved N-regions. Analysis in all 6 of these subgroups showed restriction in V(L) gene use, whereas no association between V(H) and V(L) usage was found in cases without HCDR3 similarities. Altogether, structurally similar HCDR3s associated with preferential V(L) gene usage implies selection of BCRs, especially in subsets showing high HCDR3 similarities, thus pointing to restricted antigen recognition sites and possibly involvement of specific antigens in CLL development.

Reactions of N,N-bis(2-chloroethyl)-p-aminophenylbutyric acid (chlorambucil) with 2'-deoxycytidine, 2'-deoxy-5-methylcytidine, and thymidine.

N,N-Bis(2-chloroethyl)-p-aminophenylbutyric acid (chlorambucil, 1; 2.5 mM) was allowed to react with 2'-deoxycytidine, 2'-deoxy-5-methylcytidine, and thymidine (16.1 mM) at physiological pH (cacodylic acid, 50% base), and the reactions were followed by HPLC and HPLC-MS technique. Although the predominant reaction observed was chlorambucil hydrolysis, 1 reacted with various heteroatoms of the nucleosides. The principal site of alkylation with all pyrimidine nucleosides was N3, as judged by 1H NMR and HPLC-MS analyses. Also, several other adducts were detected, which could be tentatively characterized by means of HPLC-MS and MS/MS. As expected, thymidine was the least reactive pyrimidine nucleoside studied, and in addition of the N3 derivative, it reacted only at the carbohydrate moiety. Overall reactivity of cytosine nucleosides with 1 was considerably higher. The N3 adducts of dCyd and 5-Me-dCyd partially deaminated under the reaction conditions employed, but the reaction was not catalyzed by the participation of the omega-hydroxy function of the alkyl substituent but presumably by the nitrogen atom of the chlorambucil moiety. In the case of cytosine nucleosides, the O2 derivatives were the second most abundant species. 5-Me-dCyd reacted more readily at O2 than dCyd. These O2 adducts were labile under acidic, neutral, and basic conditions. No N4 derivatives or cross-links were detected, but dCyd reacted also at C5, although the yield of this derivative was very low. The role of chlorambucil-pyrimidine 2'-deoxyribonucleoside adducts on the cytotoxicity and mutagenity of 1 is also discussed.

More extensive genetic alterations in unmutated than in hypermutated cases of chronic lymphocytic leukemia.

B-cell chronic lymphocytic leukemia (CLL) is not a uniform disease entity; approximately half of the CLL cases have undergone immunoglobulin V(H) gene hypermutation, whereas the other half display unmutated V(H) genes. We investigated genome changes in 12 hypermutated cases (M-CLL) and 22 unmutated cases (UM-CLL) by use of comparative genomic hybridization, G-banding, and multicolor fluorescence in situ hybridization (m-FISH) after optimal mitogen stimulation and FISH analysis of typical CLL aberrations: 11q deletion, 13q deletion, and trisomy 12. Very high frequencies of aberrations were found in both groups: 82% in UM-CLL and 83% in M-CLL. Deletions of 11q and 13q were equally distributed in M-CLL and UM-CLL. However, larger aberrations detectable by CGH, trisomy 12, and complex aberrations were less frequent in M-CLL than in UM-CLL. These observations led to a hypothesis that unmutated and mutated CLL have different biological Backgrounds, given that large and/or complex chromosomal aberrations and hypermutation of the CLL progenitor cells tend to be mutually exclusive.

Lymphotoxin beta expression is high in chronic lymphocytic leukemia but low in small lymphocytic lymphoma: a quantitative real-time reverse transcriptase polymerase chain reaction analysis.

BACKGROUND AND OBJECTIVES: The lymphotoxin beta (LTB) gene, localized to the major histocompatibility complex region on chromosome 6p21.3, has an important role in the formation of germinal center reactions and regulation of immune response and apoptosis. Our aim was to determine LTB gene expression in different hematologic neoplasias. DESIGN AND METHODS: We determined the expression of LTB using quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR) on a series of RNA samples from CD3(+) T cells and CD19(+) B cells isolated from peripheral blood (n=7); CD19(+) B cells isolated from lymph nodes (n=11) and from patients with acute lymphoblastic leukemia (ALL; n=16), acute myeloid leukemia (AML; n=43), chronic myeloid leukemia (CML; n=12), mantle cell lymphoma (MCL; n=19), chronic lymphocytic leukemia (CLL; n=32) and small lymphocytic lymphoma (SLL; n=22). RESULTS: The expression level of LTB in CD3(+) T cells and CD19(+) B cells isolated from blood was ten to forty times lower than that in normal CD19(+) B cells isolated from lymph nodes. In malignant myeloid cells the expression levels were very low, whereas in malignant lymphoid cells the expression was higher than in myeloid cells, being highest in MCL and CLL (20.2+/-14.0 ng/microL and 81.0+/-116.3 ng/microL) and low in SLL (4.5+/-3.6 ng/microL; p=0.001). We did not find correlations between LTB expression and hematoclinical parameters (risk groups, immunophenotypes and overall survival). INTERPRETATION AND CONCLUSIONS: A distinct difference in expression of LTB in CLL and SLL indicates that these morphologically similar B-cell malignancies are molecularly different. Further studies are needed to investigate the prognostic value of LTB and any role that LTB may have in determining whether the malignant B cells manifest a leukemia or lymphoma.

Vaccination against infections in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is a well-defined mature B-cell neoplasm associated with increased susceptibility to infections. Two major options in prevention of infections in CLL, intravenous gammaglobulin treatment and antimicrobial chemoprophylaxis, have not resulted in satisfactory outcome. A third strategy, antimicrobial vaccination, is the topic of this minireview. We collected articles and their references concerning CLL vaccination from the Medline database starting from 1966 and thirteen relevant studies were found. Plain bacterial polysaccharide vaccines would seem to be ineffective in antibody formation in patients with CLL. However, protein and conjugate vaccines appear to be more immunogenic and their responses may be further enhanced with ranitidine adjuvant treatment. New well-designed investigations are needed to develop appropriate vaccination strategies and evaluate vaccination efficacy in infection morbidity and mortality in CLL.