Perifosine treatment in chronic lymphocytic leukemia: results of a phase II clinical trial and in vitro studies.

Abstract Because of the importance of the phosphoinositide 3-kinase (PI3K)/AKT pathway in chronic lymphocytic leukemia (CLL), we evaluated in vitro cytotoxicity induced by perifosine, an AKT inhibitor, in CLL lymphocytes and found that the mean 50% effective dose (ED50) was 313 nM. We then performed a phase II trial of perifosine in patients with relapsed/refractory CLL to assess response, outcomes, toxicity and ex vivo correlative measures. After 3 months of treatment, six of eight patients showed stable disease, one achieved a partial response and one had progressive disease. Median event-free survival and overall survival in all patients treated were 3.9 and 9.7 months. Adverse events included hematologic, infectious/fever, pain, gastrointestinal and constitutional toxicities. Unexpectedly, AKT phosphorylation in CLL lymphocytes from treated patients was not correlated with response. Additionally, perifosine did not inhibit AKT phosphorylation in cultured CLL lymphocytes. Perifosine is cytotoxic to CLL cells in vitro, and largely induces stabilized disease in vivo, with an AKT-independent mechanism.

Immunogenetics shows that not all MBL are equal: the larger the clone, the more similar to CLL.

Chronic lymphocytic leukemia (CLL) -like monoclonal B-cell lymphocytosis (MBL) shares common immunophenotype and cytogenetic abnormalities with CLL, from which it is discriminated by a cutoff value of 5 × 10(9)/L circulating clonal B cells. However, the clonal size in MBL is extremely variable and allows discrimination of two distinct entities (high-count [HC] and low-count [LC]-MBL) based on a cutoff value of 0.5 × 10(9)/L clonal B cells. HC-MBL is associated with lymphocytosis and progresses to CLL requiring treatment at a rate of 1.1% per year, whereas LC-MBL is found in the general population only through high-sensitivity techniques and carries limited, if any, risk of progression. We performed an immunogenetic profiling of 333 cases with CLL-like MBL supplemented by detailed comparisons with CLL, focusing especially on CLL Rai stage 0 (CLL-0). LC- and HC-MBL had similar somatic hypermutation status, yet different IGHV gene repertoires and frequencies of B-cell receptor (BcR) stereotypy. In particular, stereotyped BcRs were infrequent in LC-MBL and were often not CLL specific. In contrast, HC-MBL exhibited clear immunogenetic similarities to CLL-0. These findings indicate that LC-MBL may not represent a true preleukemic condition, thus differing from HC-MBL/CLL-0 in which the identification of factors endowing malignant potential is strongly warranted.

Commentary: Comparison of current flow cytometry methods for monoclonal B cell lymphocytosis detection.

Monoclonal B cell lymphocytosis (MBL) is now recognized as the B-lymphocyte analogue of a monoclonal gammopathy of unknown significance. MBL can be the precursor of chronic lymphocytic leukemia or associated with non-Hodgkin's lymphoma. It may be associated with an autoimmune abnormality or be related to aging (immunosenescence). The combination of available new fluorochrome-conjugated monoclonal antibody reagents, multilaser instrumentation, and improved software tools have led to a new level of multicolor analysis of MBL. Presently, several centers, including the University of Salamanca (Spain), Duke University (Durham, NC), Mayo Clinic (Rochester, MN), and the National Cancer Institute (Bethesda, MD) in conjunction with the Genetics and Epidemiology of Familial chronic lymphocytic leukemia Consortium, the Food and Drug Administration (Bethesda, MD), and the Centers for Disease Control and Prevention/Agency for Toxic Substances and Disease Registry (Atlanta, GA) in collaboration with Saint Luke's Hospital (Kansas City, MO), the Università Vita-Salute San Raffaele in Milan (Italy), and Leeds Teaching Hospital (UK) are all actively conducting studies on MBL. This commentary is an updated summary of the current methods used in these centers. It is important to note the diversity of use in reagents, instruments, and methods of analysis. Despite this diversity, there is a consensus in what constitutes the diagnosis of MBL and its subtypes. There is also an emerging consensus on what the next investigative steps should be.

Monoclonal B cell lymphocytosis.

Monoclonal B lymphocytosis (MBL) is an asymptomatic clinical syndrome wherein small B cell clones are detectable in the peripheral blood. MBL is common in the adult population, with an estimated prevalence of greater than 3% among individuals over age 50. Most MBLs have an immunophenotype similar to chronic lymphocytic leukemia (CLL). Recently, MBL has been shown to be a precursor state for CLL, though most MBLs presumably do not progress to CLL. Therefore, there has been considerable interest in the biology of MBL to better understand the mechanisms of CLL leukemogenesis. We have investigated immunoglobulin heavy chain gene usage and clonality in MBL. These investigations reveal that most MBLs use mutated heavy chains typically associated with good-risk CLL, and that MBLs are frequently oligoclonal, rather than monoclonal. Deletion of chromosome 13q14 is also commonly observed. These and other ongoing studies may help illuminate the pathogenesis of CLL.

Malaria severity and human nitric oxide synthase type 2 (NOS2) promoter haplotypes.

Nitric oxide (NO) mediates host resistance to severe malaria and other infectious diseases. NO production and mononuclear cell expression of the NO producing enzyme-inducible nitric oxide synthase (NOS2) have been associated with protection from severe falciparum malaria. The purpose of this study was to identify single nucleotide polymorphisms (SNPs) and haplotypes in the NOS2 promoter, to identify associations of these haplotypes with malaria severity and to test the effects of these polymorphisms on promoter activity. We identified 34 SNPs in the proximal 7.3 kb region of the NOS2 promoter and inferred NOS2 promoter haplotypes based on genotyping 24 of these SNPs in a population of Tanzanian children with and without cerebral malaria. We identified 71 haplotypes; 24 of these haplotypes comprised 82% of the alleles. We determined whether NOS2 promoter haplotypes were associated with malaria severity in two groups of subjects from Dar es Salaam (N = 185 and N = 250) and in an inception cohort of children from Muheza-Tanga, Tanzania (N = 883). We did not find consistent associations of NOS2 promoter haplotypes with malaria severity or malarial anemia, although interpretation of these results was potentially limited by the sample size of each group. Furthermore, cytokine-induced NOS2 promoter activity determined using luciferase reporter constructs containing the proximal 7.3 kb region of the NOS2 promoter and the G-954C or C-1173T SNPs did not differ from NOS2 promoter constructs that lacked these polymorphisms. Taken together, these studies suggest that the relationship between NOS2 promoter polymorphisms and malaria severity is more complex than previously described.

Activation-induced cytidine deaminase expression and activity in the absence of germinal centers: insights into hyper-IgM syndrome.

Somatic hypermutation normally occurs as a consequence of the expression of activation-induced cytidine deaminase (AID) by Ag-activated, mature B cells during T cell-dependent germinal center responses. Nonetheless, despite their inability to express CD154 and initiate GC responses, patients with type 1 hyper-IgM syndrome (HIGM1) support populations of IgM(+)IgD(+)CD27(+) B cells that express mutated Ig genes. The origin of these mutated B cells is unknown; the IgM(+)IgD(+)CD27(+) cells do not express AID and appear to acquire mutations independent of stringent selection by Ag. Here, we demonstrate that immature/transitional 1 B cells from the bone marrow of CD154-deficient mice express AID and acquire Ig mutations that lack the hallmarks of antigenic selection via BCR signaling. Comparable levels of AID expression was found in developmentally immature B cells recovered from murine fetal liver and from human immature/transitional 1 B cells recovered from umbilical cord blood. AID expression in human fetal liver was also robust, approaching that of human tonsil tissue and the human germinal center B cell line, Ramos. These observations led us to conclude that AID expression in developing human B cells is the origin of the mutated IgM(+)IgD(+)CD27(+) B cells present in HIGM1 patients, and we propose that both mice and humans share a latent, AID-dependent pathway for the preimmune diversification of B lymphocytes that is more prominent in chicken, sheep, and rabbits.

Normative values for exhaled breath condensate pH and its relationship to exhaled nitric oxide in healthy African Americans.

BACKGROUND: Exhaled breath condensate (EBC) pH and exhaled nitric oxide (FeNO) have been proposed as markers of asthma severity. EBC pH values below 6.5 have been associated with asthma exacerbations. Protonation of airway nitrite occurs at low pH and may contribute to FeNO. OBJECTIVE: To establish normative EBC pH values and to determine the contribution of EBC pH to FeNO in healthy African Americans. METHODS: Two hundred seventy healthy African American subjects without asthma between 18 and 40 years old were evaluated. Subjects had simultaneous measurement of EBC pH, EBC nitrite, nitrate, and FeNO. RESULTS: The median EBC pH was 8.14 (interquartile range, 7.83-8.28). Of subjects, 11.9% had an EBC pH < or = 6.5. In subjects with EBC pH values below 6.5, there was an inverse correlation between EBC pH and FeNO (r(2) = 0.158; P = .0245; n = 32). In the entire cohort, there was a direct correlation between EBC pH and EBC nitrite (r(2) = 0.163; P < .0001), but there was no correlation between EBC nitrite and FeNO. In multivariate analyses, EBC pH and nitrite did not contribute significantly to FeNO variation while controlling for other confounders of FeNO. CONCLUSION: There was an increased prevalence (11.9%) of low EBC pH (less than 6.5) in healthy African American subjects compared with white subjects (<5%). EBC pH and nitrite were directly correlated, but there was no correlation between EBC nitrite and FeNO. FeNO correlated with EBC pH at pH values less than 6.5 in univariate but not multivariate analyses. This suggests that EBC pH and nitrite are not significant determinants of FeNO in healthy subjects.