Ibrutinib-induced lymphocytosis in patients with chronic lymphocytic leukemia: correlative analyses from a phase II study.

Ibrutinib and other targeted inhibitors of B-cell receptor signaling achieve impressive clinical results for patients with chronic lymphocytic leukemia (CLL). A treatment-induced rise in absolute lymphocyte count (ALC) has emerged as a class effect of kinase inhibitors in CLL and warrants further investigation. Here we report correlative studies in 64 patients with CLL treated with ibrutinib. We quantified tumor burden in blood, lymph nodes (LNs), spleen and bone marrow, assessed phenotypic changes of circulating cells and measured whole-blood viscosity. With just one dose of ibrutinib, the average increase in ALC was 66%, and in>40% of patients the ALC peaked within 24 h of initiating treatment. Circulating CLL cells on day 2 showed increased Ki67 and CD38 expression, indicating an efflux of tumor cells from the tissue compartments into the blood. The kinetics and degree of the treatment-induced lymphocytosis was highly variable; interestingly, in patients with a high baseline ALC the relative increase was mild and resolution rapid. After two cycles of treatment the disease burden in the LN, bone marrow and spleen decreased irrespective of the relative change in ALC. Whole-blood viscosity was dependent on both ALC and hemoglobin. No adverse events were attributed to the lymphocytosis.

Modeling tumor-host interactions of chronic lymphocytic leukemia in xenografted mice to study tumor biology and evaluate targeted therapy.

Chronic lymphocytic leukemia (CLL) cells depend on microenvironmental factors for proliferation and survival. In particular, the B-cell receptor (BCR) and nuclear factor- κB (NF-κB) pathways are activated in the lymph node (LN) microenvironment. Thus, model systems mimicking tumor-host interactions are important tools to study CLL biology and pathogenesis. We investigated whether the recently established NOD/scid/γc(null) (NSG) mouse xenograft model can recapitulate the effects of the human microenvironment. We assessed, therefore, tumor characteristics previously defined in LN-resident CLL cells, including proliferation, and activation of the BCR and NF-κB pathways. We found that the murine spleen (SP) microenvironment supported CLL cell proliferation and activation to a similar degree than the human LN, including induction of BCR and NF-κB signaling in the xenografted cells. Next, we used this model to study ibrutinib, a Bruton's tyrosine kinase inhibitor in clinical development. Ibrutinib inhibited BCR and NF-κB signaling induced by the microenvironment, decreased proliferation, induced apoptosis and reduced the tumor burden in vivo. Thus, our data demonstrate that the SP of xenografted NSG mice can, in part, recapitulate the role of the human LN for CLL cells. In addition, we show that ibrutinib effectively disrupts tumor-host interactions essential for CLL cell proliferation and survival in vivo.

[A 62 year old woman with spontaneous onset of distinct walking disorder and pancytopenia]. / Eine 62-jährige Frau mit progredienter Gangstörung und Panzytopenie.

Our case-report presents a 62 year old woman with spontaneous onset of a distinct walking disorder and pancytopenia. The diagnosis showed a sub-acute combined degeneration of the spinal cord (SACD) and a megaloblastic anemia as a result of a cobalamin deficiency. This was caused by a strict vegetarian nutrition since 18 years. Once on therapy with cobalamin substitutes the clinical and laboratory test results improved within 3 months.

Telomerase activity in skeletal sarcomas.

BACKGROUND: Telomerase is a ribonucleoprotein that adds TTAGGG nucleotide repeats onto the ends of eukaryotic chromosomes to maintain telomere integrity. Somatic cells do not express telomerase and stop dividing when the chromosomal ends are shortened critically after many cell divisions. Immortal cell lines and cancer cells apparently have telomerase activity that contributes to an unlimited number of cell cycles. The purpose of our study is to investigate whether telomerase activity is expressed in primary malignant tumors of the skeletal system when compared to adjacent normal tissue. METHODS: Fresh tumor and normal tissue was collected from 14 patients (10 males, 4 females; age range, 8 to 76 years) and protein extraction performed. The tumors included seven osteosarcomas (three examined before and after chemotherapy), two chondrosarcomas, two spindle cell tumors, one hemangiopericytoma, one chordoma, and one adamantinoma. Telomerase activity was analyzed by using a highly sensitive polymerase chain reaction (PCR)-based assay (telomere repeat amplification protocol [TRAP]). RESULTS: Telomerase activity was found in 8 of 14 sarcoma patients (57%) using the TRAP assay. Compared to HeLa cell extract (positive control), telomerase activity in the tumor specimen ranged from 0 (in osteosarcoma) to 11.7% (in hemangiopericytoma). There was variation in the number of telomeric repeats generated by telomerase. At least five telomeric bands (e.g. 50, 56, 62, 68, 74 bp) in a ladder pattern had to be present before telomerase activity was considered positive in our analysis. CONCLUSIONS: Telomerase activity may be an oncogenic sustaining event helping to maintain the transformed phenotype seen in malignant tumors of the bone. The degree of telomerase activity varies among skeletal malignancies, but was less than that observed in HeLa cells. The majority of osteosarcomas showed no telomerase activity.

Comparison of chromosome telomere integrity in multiple tissues from subjects at different ages.

Telomere DNA, at the ends of each chromosome, is conserved in nature and required for chromosome replication and stability. Reduction in telomere length has been observed in several malignancies as well as in leukocytes from healthy persons with advancing age. There is a paucity of data regarding telomere length and the effects of in vivo aging in different tissues. These data could be helpful in interpreting telomere length and understanding the role of telomere integrity and telomerase activity in malignant cells. We report telomeric DNA integrity studies of blood and skin collected from eight Caucasians of both sexes representing each decade of life from the fetus to 72 years of age without exposure to chemotherapy or radiation. In addition, telomeric data from 15 other tissues from the fetus and 8 other tissues from the 72-year-old male were examined. No significant differences were found in the shortest telomere size, the average telomere size, or telomere size variation between blood and skin from subjects at different ages. The average telomere size was 11.7 +/- 2.2 kb for blood and 12.8 +/- 3.7 for skin in all subjects studied. The shortest telomere length was 5.4 +/- 1.9 kb for blood and 4.3 +/- 0.9 kb for skin. Significant differences (P < 0.001) were found in the overall length of the DNA hybridization signal representing the shortest telomere size and the length of the DNA peak migration hybridization signal representing variation in telomere size between the 20-week fetus and the 72-year-old male. The 72-year-old male showed the shortest telomeres and the most variation (heterogeneity) in telomere size for all tissues studied, but the greatest differences were observed in blood compared with other tissues (e.g., average telomere length was 12.2 kb in the fetus and 7.2 kb in the 72-year-old male). The size of the telomere was negatively correlated with age for all tissues studied.

Clear cell sarcoma or malignant melanoma of soft parts: molecular analysis of microsatellite instability with clinical correlation.

Malignant melanoma of soft parts, also termed clear cell sarcoma (CCS), is a rare malignancy of neural crest origin which is different from cutaneous malignant melanoma. Although a translocation involving chromosomes 12 and 22 is characteristic of clear cell sarcoma and not malignant melanoma, there are a paucity of methods to differentiate the two. Therefore, a study of microsatellite instability (MIN) was undertaken to determine if mechanisms of DNA mismatch repair can differentiate these malignancies. MIN has been described in a variety of malignancies including 25% of malignant melanomas. Paraffin-embedded neoplastic and non-neoplastic cells were obtained from 11 individuals (five males; six females; age range from seven to 60 years) with CCS. Isolated DNA was PCR amplified at 17 separate microsatellite loci using radioactive-labeled primers. Tumor tissue was compared to normal tissue for each analysis. No MIN was detected. Loss of heterozygosity was detected in only one patient at a single locus (IFNA). The lack of MIN in clear cell sarcoma further defines the distinction between this tumor and malignant melanoma. Clinically, local recurrence and metastasis were indicators of poor outcome. The size of the tumor was not a significant prognostic indicator. Local recurrence, satellitosis, or nodal metastasis was not proven to be uniformly fatal. Utilization of chemotherapy and/or radiation demonstrated no obvious survival advantage. The histologic parameters of mitotic rate and the presence of necrosis were not prognostic. Limb-preserving surgical procedures were as effective as amputation for local disease control. The actuarial survival rate was calculated to be 48% at five years.

Quantitative evaluation of the beta 2-adrenoceptor affinity of phenoxypropanolamines and phenylethanolamines.

The influence of the aromatic moiety of beta-adrenoceptor ligands on the affinity for the beta 2-adrenoceptor has been studied. Three classes of ligands have been examined, viz. N-isopropyl- and N-tert-butylphenylethanolamines and N-isopropylphenoxypropanolamines. Computer-assisted analysis of the inhibition by any of these ligands of the specific (-)-[3H]dihydroalprenolol binding to the beta 2-adrenoceptors of a bovine skeletal muscle preparation in the presence of GppNHp (10(-4) M) yielded the affinities of these ligands at pH 7.5. The obtained values were adjusted for the amounts of cations present at this pH value. A significant correlation was found between the calculated lipophilicities and the experimentally determined affinities in the three classes. Furthermore, steric factors seem to play an important role, as these correlations were improved by the introduction of steric parameters for the aromatic substituents in the regression analyses. From the established equations it is concluded that the phenoxypropanolamine derivatives bind to the beta 2-adrenoceptor in a way different from that of the ligands in both ethanolamine classes.

Factors controlling beta 1-adrenoceptor affinity and selectivity.

A membrane preparation of the calf heart left ventricle has been used after identification and characterization, as a source of myocardial beta 1-adrenoceptor for radioligand binding studies. The displacement of specifically bound (-)-[3H]dihydroalprenolol by some beta-adrenoceptor ligands appeared to be pH-dependent, which could be related to the ionization characteristics of the compounds. Among the usually four ionic species of the ligand, present at physiological pH, the cation was shown to govern beta 1-adrenoceptor affinity. Furthermore, quantitative structure affinity relationships for the interaction with beta 1- and beta 2-adrenoceptors were established for the phenoxypropanolamines, a class of beta-adrenoceptor ligands. The N-isopropyl-oxypropanolamine side chain itself does not discriminate between beta 1- and beta 2-adrenoceptors, whereas aromatic substitution ortho to the side chain induces some beta 2-selectivity. Selectivity for myocardial beta 1-adrenoceptors is mainly obtained by aromatic substitution para to the side chain. This substitution pattern yields a decrease in beta 2-adrenoceptor affinity, far more pronounced than the decrease in beta 1-adrenoceptor affinity.