Epigenetic mechanisms of protein tyrosine phosphatase 6 suppression in diffuse large B-cell lymphoma: implications for epigenetic therapy.

Protein tyrosine phosphatases such as PTPN6 can be downregulated in various neoplasms. PTPN6 expression by immunohistochemistry in 40 diffuse large B-cell lymphoma (DLBCL) tumors was lost or suppressed in 53% (21/40). To elucidate the molecular mechanisms of PTPN6 suppression, we performed a comprehensive epigenetic analysis of PTPN6 promoter 2 (P2). None of the DLBCL primary tumors (0/37) had PTPN6 hypermethylation on the CpG1 island using methylation-specific PCR, pyrosequencing, and high-resolution melting assays. However, hypermethylation in 57% (21/37) of cases was found in a novel CpG island (CpG2) in P2. PTPN6 gene suppression was reversed by 5-aza-deoxycytidine (5-Aza), a DNA methyltransferase inhibitor, and the histone deacetylase inhibitor (HDACi) LBH589. LBH589 and 5-Aza in combination inhibited DLBCL survival and PTPN6 hypermethylation at CpG2. The role of histone modifications was investigated with a chromatin-immunoprecipitation assay demonstrating that PTPN6 P2 is associated with silencing histone marks H3K27me3 and H3K9me3 in DLBCL cells but not normal B cells. 3-Deazaneplanocin A, a histone methyltransferase inhibitor, decreased the H3K27me3 mark, whereas HDACi LBH589 increased the H3K9Ac mark within P2 resulting in re-expression of PTPN6. These studies have uncovered novel epigenetic mechanisms of PTPN6 suppression and suggest that PTPN6 may be a potential target of epigenetic therapy in DLBCL.

Analysis of clonal B-cell CD38 and immunoglobulin variable region sequence status in relation to clinical outcome for B-chronic lymphocytic leukaemia.

Recent reports suggest that the expression of germline (GL) Ig variable region heavy-chain genes (VH) is a negative prognostic factor for B-cell chronic lymphocytic leukaemia (B-CLL) patients and that CLL B-cell CD38 expression may be a surrogate marker of Ig VH gene status. Currently, however, the usefulness of this surrogate marker is controversial. Therefore, our goal was to study the ability of CD38 to act as a surrogate marker for Ig VH somatic mutation (SM), and to identify differences in overall survival (OS), progression-free survival (PFS) and response in B-CLL patients based on these two markers. We first assessed the relationship between CD38 expression and Ig VH status on 131 B-CLL patients, including 66 patients enrolled in three North Central Cancer Treatment Group Trials. Although the mean percentages of CD38+ clonal B cells were significantly higher for patients classified as GL versus SM, CD38 was not a reliable marker for clonal B-cell SM. Overall, GL patients exhibited significantly shorter OS and PFS times than SM patients. Despite the inability of clonal B-cell CD38 expression to predict Ig VH mutation status, patients with < or =30% CD38+ cells did have shorter PFS and OS times than did CLL patients with < 30% CD38+ cells. Thus, the relationship between CD38 expression and Ig VH mutation status in B-CLL is not straightforward. Nevertheless, analysis in a co-operative group clinical trial setting suggests that both B-cell markers alone or in combination may have clinical usefulness. These data strongly encourage the study of these biological markers as they relate to disease heterogeneity in B-CLL.

S-phase fraction by the labeling index as a predictive factor for progression and survival in low grade non-Hodgkin's lymphoma.

BACKGROUND: The purpose of this study was to learn if the bromodeoxyuridine labeling index (LI), a measure of the S-phase fraction, is an independent prognostic factor for overall survival (OS) for patients with newly diagnosed low grade non-Hodgkin's lymphoma (NHL). In addition, the ability of the LI to predict time to progression (TTP) in a group of patients observed without therapy after initial diagnosis was determined. METHODS: Patients eligible for this study had biopsy proven low grade NHL, adequate tissue to perform the LI, and were previously untreated. The bromodeoxyuridine LI was performed on fresh biopsy samples using a slide-based immunofluorescence procedure. RESULTS: One-hundred twelve patients were followed prospectively for OS, and 50 of these patients who initially were observed without therapy were eligible for an analysis of TTP. The LI (< or = 1% vs. > 1%) and presence of "B" symptoms were significant univariate prognostic factors for survival (P values of 0.004 and < 0.001, respectively). In a multivariate analysis, the LI and symptoms retained independent prognostic significance, whereas disease stage, histologic subtype, and age did not. In the group who were observed after diagnosis, the LI was not an independent predictor of TTP. CONCLUSIONS: The LI at initial diagnosis is an independent prognostic factor for OS of patients with low grade NHL, but it does not help choose patients for observation without therapy. Measurements of the LI should be considered as part of the on-study evaluation of patients entering cooperative group trials evaluating new therapies for this group of lymphomas.

Effects of phenylacetate on cells from patients with B-chronic lymphocytic leukemia.

Peripheral blood mononuclear cells from 11 patients with untreated B-chronic lymphocytic leukemia (CLL) were exposed to sodium phenylacetate (NaPA) in culture to assess its ability to induce differentiation. We found no evidence of cellular differentiation or induction of tartrate resistant acid phosphatase activity, as seen when B-CLL cells were treated with phorbol ester. We observed a striking decrease in the viability of the B-CLL cells in a time and dose dependent fashion when exposed to NaPA. After six days of culture, control cells from the 11 patients studied had a median viability of 90%, whereas cells exposed to NaPA at 5 and 10 mM concentrations had median viabilities of 39 and 16%, respectively. The cells treated with NaPA developed prominent cytoplasmic vacuoles. NaPA binds and depletes glutamine which is an important amino acid for lymphocyte metabolism. Although the mechanism of the cytocidal effects demonstrated in this study are unknown, they may relate at least partially to glutamine deprivation.

Models of S-phase determination in lymphomas from flow cytometric DNA content histograms: comparison with the bromodeoxyuridine labeling index.

The estimated S-phase fraction (%S) of non-Hodgkin's lymphomas has been demonstrated to be of prognostic value. The %S can be determined by labeling index (LI) techniques or by various mathematical models applied to DNA content histograms. We performed DNA content analysis and a slide-based bromodeoxyuridine immunofluorescence LI on split samples from 117 biopsy specimens suspicious for lymphoma. The LI was compared with the %S determined by two computer models (rectangular and polynomial) with and without debris subtraction, and a manual gates computer model. In the 93 DNA diploid cases, the rectangular models had the highest correlation with the LI (R = 0.88). In the 24 aneuploid cases, the manual gates model was the most useful because of a high correlation with the LI (R = 0.70) and its ability to be used in most cases (23/24). The polynomial models had limited usefulness because they generally gave a higher %S than the LI and could be fitted in less than half of the DNA aneuploid histograms. These results suggest that in situations where the LI is not possible, an accurate %S estimate can usually be obtained with either a rectangular or manual gates model.

Rapid immunotyping of B-cell non-Hodgkin's lymphomas by flow cytometry. A comparison with the standard frozen-section method.

The authors compared immunotyping (IT) results obtained by both standard frozen section (FS) and flow cytometry (FC) methods on 218 biopsies suggestive of lymphoma to learn the advantages of each method. The independent interpretations of the FS and FC IT results were concordant in 93% (202 of 218) of cases. The 16 cases with discordance were reviewed and seven causes for discrepancy found: methodologic problems, focal lymphomatous involvement, more sensitive light chain detection by FC, inadequate sample for FC, interpretation error, sample mislabeling for FC, and unexplained. Eleven of the concordant B-cell non-Hodgkin's lymphomas (NHLs) studied by FC did not have a kappa:lambda ratio of 3 or greater or 0.5 or less and were shown to express light chain restriction by a D-value of 15 or greater with the use of statistical analysis of the kappa and lambda histograms or by multiparameter analysis of large versus small cells. The authors found both methods to be effective for phenotyping lymphomas, however, each has distinct features, making them complementary in their applications.

Rapid S-phase determination of non-Hodgkin's lymphomas with the use of an immunofluorescence bromodeoxyuridine labeling index procedure.

Cell kinetic measurements are currently being investigated to determine if they are useful in the clinical management of patients with non-Hodgkin's lymphomas (NHLs). Although the tritiated thymidine labeling index (TLI) is the standard method of S-phase measurement, it is difficult to perform. The authors describe a slide-based immunofluorescence labeling index (LI) method that uses 5-bromo-2-deoxyuridine (BrdUrd) as the pulsing medium and a monoclonal antibody (BU-1) to BrdUrd. The BrdUrd LI was performed on 217 NHLs and compared with routine histologic results. The authors found a median BrdUrd LI of 0.9% for low-grade NHLs; 7.5% for intermediate-grade; 10.4% for high-grade; and 2.2% for T-cell NHLs. This method provides a rapid, reliable S-phase measurement that can be easily performed in the clinical laboratory. It should replace the TLI and allow wider application of S-phase measurements in the NHL.

Evaluation of the AnIdent system for the identification of anaerobic bacteria.

AnIdent is a new 4-hr system for the identification of anaerobic bacteria that depends upon detection of a unique set of preformed enzymes. A single AnIdent test speciated 76% of 333 anaerobes (89% agreement with a conventional system used at the Mayo Anaerobe Laboratory) without any repeat or supplemental testing (level 1). Additional testing increased the identification rate to 93% (level 2). We conclude that AnIdent is a reliable method for identifying clinically significant anaerobes which only occasionally requires the use of a small number of simple supplemental tests and avoids the necessity for performing gas-liquid chromatography.