ALBERT

Description: The International Prognostic Factor Index (IPI) predicts survival in DLBCL in patients treated with chemotherapy. The Revised IPI (R-IPI) has been reported to be a simpler and more accurate predictor of outcome in patients treated with immunochemotherapy (rituximab and anthracycline-based chemotherapy). We evaluated the predictive value of the IPI and the R-IPI in an observational cohort of unselected patients treated with R-CHOP. Consecutive, newly diagnosed patients age 18 years and older with DLBCL were prospectively offered enrollment into our Lymphoma SPORE Registry. Pathology was centrally reviewed, and composite lymphomas and history of concurrent or prior cancers were excluded. All patients were actively followed for progression free progression (PFS) and overall survival (OS). Here we report on patients enrolled from 9/2002 – 6/2006. 229 patients with a median age of 62 years (range 20–93) were evaluated. 56% were 〉60 years of age, 16% had a performance score ≥2, 54% had an elevated LDH, 19% had 〉1 extranodal site, and 51% were stage III/IV. During follow-up, there were 63 progressions (28%) and 45 deaths (20%), and the median follow-up time for living patients was 34 months (range 6–61 months). As shown in the table and figure, the IPI and R-IPI were predictive for both PFS and OS (all p

Description: Background: Patients with CLL have a variable clinical course. Current novel prognostic indicators (ZAP-70, CD38, IgVH gene mutation status, FISH) are able to identify early-stage CLL patients at high risk of rapid disease progression. These prognostic factors focus on characteristics of the malignant B cell clone and are not currently changeable factors. Identification of modifiable characteristics of the host unrelated to the leukemic B-cell but that relate to CLL-specific survival may provide opportunities for therapeutic intervention. Since the immune system is likely to modulate disease progression in CLL patients, we evaluated critical features of that system in relation to the size of the circulating clonal B-cell population in a CLL cohort. Methods: 186 consecutive patients with CLL who were evaluated at Mayo Clinic within 2 months of diagnosis between 2000 and 2002 were identified. The primary end-point of the study was to assess if host immunity affected time to treatment (TTT). Baseline flow cytometry analysis was available for 166 patients (88%) and was used to calculate the absolute number of T-cell and natural kill (NK)-cells present at diagnosis. The size of the T-cell/NK-cell compartment relative to the size of the malignant monoclonal B-cell (MBC) compartment was then evaluated by calculating the NK:MBC ratio and T:MBC ratio. Relationships with other prognostic parameters and with TTT were evaluated. Results: Patients exhibited substantial variation in the absolute number of T-cells and NK cells as well as T:MBC and NK:MBC ratios at the time of diagnosis. Higher T:MBC and NK:MBC ratios were observed among patients with early Rai stage and mutated IgVH genes (all P≤0.0002). As continuous variables, both the T:MBC ratio (p value=0.03) and NK:MBC ratio (p value=0.02) were associated with TTT. Since they correlated with TTT as continuous variables, thresholds to classify MBC, T:MBC ratio, and NK:MBC were identified using the method of Contal and O’Quigly. On multivariate Cox modeling including stage, CD38, absolute MBC count, T:MB ratio, and NK:MB ratio, the independent predictors of TTT were disease stage, T:MB ratio, and NK:MB ratio (Table 1). Conclusion: Measurable characteristics of the host immune system appear to relate to the rate of disease progression in patients with newly diagnosed CLL. These characteristics can be modified and continued evaluation of immunomodulatory drugs, vaccination strategies, and cellular therapies to delay/prevent disease progression are warranted. Additional studies exploring how interactions between the host immune system and the leukemic clone influence clinical outcomes are needed.

Description: Background: CLL is still an incurable lymphoid malignancy. The current standard of care is to treat only patients with obvious clinical progression as defined by the National Cancer Institute Working Group in 1996 (NCI–WG 1996). High risk CLL can be identified at diagnosis by a variety of tests including fluorescence in situ hybridization (FISH), immunoglobulin heavy-chain variable region (IgVH) analysis, and expression of ZAP-70 or CD38. With this information we can identify patients with high risk, early stage disease who are candidates for novel low toxicity therapies that could alter the natural course of their disease. Our hypothesis was that combination monoclonal antibody (MoAb) therapy using alemtuzumab and rituximab would significantly reduce the high risk clone in early stage CLL. Methods: This phase II trial was conducted with IRB approval and accrued the planned 30 patients between January 2005 and July 2007 at Mayo Clinic Rochester. The study enrolled consenting patients with Rai stage 0–II CLL without NCI–WG 1996 criteria for treatment who had high risk CLL as defined as one or more of the following 17p13–, 11q22–, unmutated (UM) IgVH (〈 2%) and expression of ZAP–70 (≥ 30%) and/or CD38 (≥20%). Treatment was one 30 day cycle (alemtuzumab 3 mg, 10 mg, 30 mg days 1–3 then 30 mg 3 × week × 4 weeks with all doses subcutaneously and rituximab at 375 mg/m2/week IV × 4 doses from day 8). Patients received 7 days of allopurinol and antimicrobial prophylaxis against PCP and herpes virus infections for 7 months. CMV testing by PCR was done weekly during treatment, then monthly × 6. Results: All 30 patients have completed therapy and 27 have been evaluated for response. The median age of these 27 patients was 62 yr (range 29–77) with 8 patients 〉 70 years. There was a male predominance (67%) with median time from diagnosis to treatment of 0.7 yr (range 0.1 – 6.1). Stage (Rai) at the start of therapy was 0 in 7 (26%), I in 19 (70%), and II in 1. High risk markers were 17p13– in 9 (33%), 11q22– in 7 (26%), UM IgV and expression of either ZAP–70 or CD38 in 11 (41%). All 27 patients completed therapy without interruption. Non hematological grade 3–4 toxicity occurred in 3 patients (2 drug reactions caused by SMX/TMP, 1 CMV reactivation responsive to IV therapy). Response evaluation at 2 months after completion of therapy using NCI–WG 1996 criteria showed an ORR of 93% with 12 (44%) CR, 8 (30%) nPR, and 5 (19%) PR. Two (7%) patients had disease progression. Median duration of follow up was 14.1 months (range 1.9 – 27.2). Median time to disease progression in the 25 responding patients was 14.4 months (95% CI 5.9 – 22.4). Seven (26%) patients have received subsequent treatment for CLL (median 5.2 months, range 2.4 – 20.1). A minimal residual disease assay using 3-color flow cytometry on peripheral blood was negative in 6 of the 7 patients with CR who had no evidence of residual CLL on immunohistochemical examination of the bone marrow. These 6 patients all remain in sustained CR (median follow up 15 months, range 2 – 27.2). Conclusion: Alemtuzumab and rituximab is an effective and tolerable therapy in patients with high risk early stage CLL. A randomized controlled trial is now required to test if this intervention is better than the standard observation approach. In addition, this combination MoAb regimen could be used as a platform to develop even more effective combination treatments for patients with CLL.

Description: Recognition of pathogen-derived carbohydrate constituents by antigen presenting cells is an important step in the induction of protective immunity. Here we investigated the interaction of L-SIGN (liver/lymph node specific ICAM-3-grabbing nonintegrin), a C-type lectin that functions as antigen receptor on human liver sinusoidal endothelial cells, with egg-derived glycan antigens of the parasitic trematode Schistosoma mansoni. Our data demonstrate that L-SIGN binds both schistosomal soluble egg antigens (SEA) and egg glycosphingolipids, and can mediate internalization of SEA by L-SIGN expressing cells. Binding and internalization of SEA was strongly reduced after treatment of SEA with endoglycosidase H, whereas defucosylation affected neither binding nor internalization. These data indicate that L-SIGN predominantly interacts with oligomannosidic N-glycans of SEA. In contrast, binding to egg glycosphingolipids was completely abolished after defucosylation. Our data show that L-SIGN binds to a glycosphingolipid fraction containing fucosylated species with compositions of Hex1HexNAc5−7dHex3−6Cer, as evidenced by mass spectrometry. The L-SIGN “gain of function” mutant Ser363Val, which binds fucosylated Lewis antigens, did not bind to this fucosylated egg glycosphingolipid fraction, suggesting that L-SIGN displays different modes in binding fucoses of egg glycosphingolipids and Lewis antigens, respectively. Molecular modeling studies indicate that the preferred binding mode of L-SIGN to the respective fucosylated egg glycosphingolipid oligosaccharides involves a Fucα1-3GalNAcβ1-4(Fucα1-3)GlcNAc tetrasaccharide at the nonreducing end. In conclusion, our data indicate that L-SIGN recognizes both oligomannosidic N-glycans and multiply fucosylated carbohydrate motifs within Schistosoma egg antigens, which demonstrates that L-SIGN has a broad but specific glycan recognition profile.

Description: Recent gene-expression data have suggested that host immune genetic signatures may predict outcomes in patients with follicular lymphoma. We evaluated the hypothesis that germ line common variation in candidate immune genes is associated with survival. Cox models were used to estimate hazard ratios (HR) and corresponding 95% confidence intervals for individual SNPs after accounting for age, clinical, and other demographic factors. The median age at diagnosis of the 278 patients was 57 years, and 59 (21%) of the patients died during follow-up, with a median follow-up of 59 months (range, 27-78 months) for surviving patients. SNPs in IL8 (rs4073; HRTT = 2.14, 1.26-3.63), IL2 (rs2069762; HRGT/TT = 1.80, 1.06-3.05), IL12B (rs3212227; HRAC/CC = 1.83, 1.06-3.06), and IL1RN (rs454078; HRAA = 1.93, 1.11-3.34) were the most robust predictors of survival. A summary score of the number of deleterious genotypes from these genes was strongly associated with survival (P = .001). A risk score that combined the 4 SNPs with the clinical and demographic factors was even more strongly associated with survival (P 〈 .001); the 5-year Kaplan-Meier survival estimates were 96% (93%-100%), 72% (62%-83%), and 58% (48%-72%) for groups at low, intermediate, and high risk, respectively. Common variation in host immune genes warrants further evaluation as a promising class of prognostic factors in follicular lymphoma.

Description: BCR-ABL is responsible for the initial phase of chronic myelogenous leukemia (CML), which is effectively treated by the BCR-ABL tyrosine kinase inhibitor imatinib. Over time, patients become resistant to treatment and progress to blast crisis, an event that is driven by additional genetic and epigenetic aberrations. Recently, we showed that RIZ1 expression decreases in blast crisis and that re-expression of RIZ1 inhibits IGF-1 expression. IGF-1 signaling is required in many stages of hematopoiesis and inappropriate activation of autocrine IGF-1 signaling may facilitate transformation to blast crisis. In this study, we examined mechanisms used by CML blast crisis cells to activate IGF-1 expression. We monitored the effects of small molecule inhibitors and shRNAs of BCR-ABL, HCK, and STAT5b on IGF-1 expression, proliferation, and apoptosis in CML blast crisis cell lines. We used chromatin immunoprecipitation assays to confirm that STAT5b associates with the IGF-1 promoter in a BCR-ABL-dependent manner. We found that BCR-ABL activates autocrine IGF-1 signaling using HCK and STAT5b. Imatinib blocks HCK phosphorylation, STAT5b phosphorylation, and the association of STAT5b with the IGF-1 promoter. Inhibition of these signaling components using small molecule drugs or shRNA inhibits IGF-1 expression, decreases proliferation, and enhances apoptosis. The effects of imatinib are partially reversed by the addition of exogenous IGF-1. Furthermore, treatment of CML blast crisis cell lines with the IGF-1 receptor inhibitor AG1024 decreases proliferation and enhances apoptosis and this activity correlates with IGF-1 expression levels. Our study highlights autocrine IGF-1 signaling as an important event in the transformation to blast crisis and provides a novel mechanism to explain the activity of IGF-1R and HCK inhibitors in blocking blast crisis phenotypes. Together, our results provide further support for the therapeutic potential of IGF-1 signaling components in treating CML, especially in cases associated with elevated autocrine IGF-1 expression.

Description: Re-expression of hypermethylated tumor suppressor genes using epigenetic modifiers, such as DNA methyltransferase (DNMT) and histone deacetylase (HDAC) inhibitors, occurs by a mechanism whereby promoter demethylation is the dominant event. In support of this model, we found that the DNMT inhibitor 5-Aza-2-deoxycytidine (decitabine) induces expression of the tumor suppressor gene p15INK4B (p15) in AML cells with hypermethylated p15 promoters. Re-expression of p15 by decitabine is associated with decreases in p15 promoter methylation and histone H3 lysine 9 (H3K9) methylation and increases in H3K9 acetylation. DNA methylation is linked to H3K9 methylation through the DNA methyl binding protein MeCP2, which associates with DNMTs and H3K9 methyltransferases. Using chromatin immunoprecipitaton (ChIP) assays, we confirmed that MeCP2, DNMT1 and the H3K9 methylatransferase SUV39H1 interact with the methylated p15 promoter and that this interaction is reduced by decitabine. To determine whether promoter demethylation is also dominant to H3K9 demethylation, we monitored p15 re-expression in the presence of SUV39H1 shRNA alone and in combination with decitabine. SUV39H1 shRNA induces p15 expression and H3K9 demethylation, however it does not affect p15 promoter methylation. These results are in contrast to the HDAC inhibitor trichostatin A (TSA), which cannot induce p15 re-expression. SUV39H1 shRNA induced p15 expression and H3K9 demethylation are also enhanced by co-treatment with decitabine or TSA. Surprisingly, co-treatment with decitabine and SUV39H1 shRNA partially reverses decitabine induced promoter demethylation. Using ChIP assays we show that SUV39H1 shRNA increases the amount of the histone H3K9 dimethytransferase G9a and DNMT1 associated with the p15 promoter. Increased levels of G9a at the p15 promoter would enhance promoter methylation since G9a stimulates DNMT1 activity. Our results demonstrate that hypermethylated p15 can be reactivated in AML cells by an initial event that involves H3K9 demethylation. In addition, we found that the SUV39H1 inhibitor chaetocin induces p15 in AML cells with hypermethylated p15 promoters. Therefore, H3K9 methylatransferases represent novel therapeutic targets for developing inhibitors to reactivate the expression of hypermethylated genes.