Species D Adenoviruses as Oncolytics against B-cell Cancers

Purpose: Oncolytic viruses are self-amplifying anticancer agents that make use of the natural ability of viruses to kill cells. Adenovirus serotype 5 (Ad5) has been extensively tested against solid cancers, but less so against B-cell cancers because these cells do not generally express the coxsackie and adenoviral receptor (CAR). To determine whether other adenoviruses might have better potency, we mined the adenovirus virome of 55 serotypes for viruses that could kill B-cell cancers. Experimental Design: Fifteen adenoviruses selected to represent Ad species B, C, D, E, and F were tested in vitro against cell lines and primary patient B-cell cancers for their ability to infect, replicate in, and kill these cells. Select viruses were also tested against B-cell cancer xenografts in immunodeficient mice. Results: Species D adenoviruses mediated most robust killing against a range of B-cell cancer cell lines, against primary patient marginal zone lymphoma cells, and against primary patient CD138þ myeloma cells in vitro. When injected into xenografts in vivo, single treatment with select species D viruses Ad26 and Ad45 delayed lymphoma growth. Conclusions: Relatively unstudied species Dadenoviruses have a unique ability to infect and replicate in B-cell cancers as compared with other adenovirus species. These data suggest these viruses have unique biology in B cells and support translation of novel species D adenoviruses as oncolytics against B-cell cancers

Differentiation of Chronic Lymphocytic Leukemia B Cells into Immunoglobulin Secreting Cells Decreases LEF-1 Expression

Lymphocyte enhancer binding factor 1 (LEF-1) plays a crucial role in B lineage development and is only expressed in B cell precursors as B cell differentiation into mature B and plasma cells silences its expression. Chronic lymphocytic leukemia (CLL) cells aberrantly express LEF-1 and its expression is required for cellular survival. We hypothesized that modification of the differentiation status of CLL cells would result in loss of LEF-1 expression and eliminate the survival advantage provided by its aberrant expression. In this study, we first established a methodology that induces CLL cells to differentiate into immunoglobulin (Ig) secreting cells (ISC) using the TLR9 agonist, CpG, together with cytokines (CpG/c). CpG/c stimulation resulted in dramatic CLL cell phenotypic and morphologic changes, expression of cytoplasmic Ig, and secretion of light chain restricted Ig. CpG/c stimulation also resulted in decreased CLL cell LEF-1 expression and increased Blimp-1 expression, which is crucial for plasma cell differentiation. Further, Wnt pathway activation and cellular survival were impaired in differentiated CLL cells compared to undifferentiated CLL cells. These data support the notion that CLL can differentiate into ISC and that this triggers decreased leukemic cell survival secondary to the down regulation of LEF-1 and decreased Wnt pathway activation

Selective Induction of DNA Repair Pathways in Human B Cells Activated by CD4+ T Cells

Greater than 75% of all hematologic malignancies derive from germinal center (GC) or post-GC B cells, suggesting that the GC reaction predisposes B cells to tumorigenesis. Because GC B cells acquire expression of the highly mutagenic enzyme activation-induced cytidine deaminase (AID), GC B cells may require additional DNA repair capacity. The goal of this study was to investigate whether normal human B cells acquire enhanced expression of DNA repair factors upon AID induction. We first demonstrated that several DNA mismatch repair, homologous recombination, base excision repair, and ATR signaling genes were overexpressed in GC B cells relative to naïve and memory B cells, reflecting activation of a process we have termed somatic hyperrepair (SHR). Using an in vitro system, we next characterized activation signals required to induce AID expression and SHR. Although AID expression was induced by a variety of polyclonal activators, SHR induction strictly required signals provided by contact with activated CD4+ T cells, and B cells activated in this manner displayed reduced levels of DNA damage-induced apoptosis. We further show the induction of SHR is independent of AID expression, as GC B cells from AID -/- mice retained heightened expression of SHR proteins. In consideration of the critical role that CD4+ T cells play in inducing the SHR process, our data suggest a novel role for CD4+ T cells in the tumor suppression of GC/post-GC B cells

2009

ABSTRACT to evaluate the experience of CLL patients who were ≤55 years of age at the time of diagnosis. The threshold of ≤55 years was selected to classify age based on a similar cutoff used to define young CLL patients in historical studies. Statistical analysis Patients' characteristics are summarized using medians and ranges for continuous variables and frequencies (percentages) for categorical variables. Overall survival was defined as time from diagnosis to date of death due to any cause or last follow-up time, whichever occurred first. Time to first treatment was defined as the time between the date of diagnosis and the date of initiation of treatment or date of last follow-up at which the patient was known to be untreated. Since the indications for treatment under both the 1996 38 P values <0.05 were considered statistically significant. All statistical analyses were conducted using the SAS 9.2 software package (SAS Institute, Cary, NC, USA). More details on the methods and statistical analysis can be found in the Online Supplementary Appendix. Results Baseline characteristics of patients ≤ 55 years of age relative to those >55 years old Between January 1, 1995 and April 9, 2012, 844 patients ≤55 years old (median age, 50 years) with CLL were seen at the Mayo Clinic. A comparison of the baseline characteristics and prognostic profile of these patients relative to the 2324 CLL patients >55 years old (median age, 67 years) seen at the Mayo Clinic during the same time period is shown in We next divided patients ≤aged 55 into three groups based on their age at diagnosis: (i) age ≤45 years (n=204, 24%), (ii) age 46-50 years (n=272, 32%) and (iii) age 51-55 years (n= 368, 44%). Patients ≤45 years were more likely to present with intermediate (I or II) and high (III and IV) Rai risk (P=0.009) and be IGHV unmutated (P=0.03) compared to those in the groups 46-50 or 51-55 years old (Online After a median follow-up of 5.5 years (range, 0-17 years), 426/844 (51%) patients ≤ 55 years old had been treated. Substantial changes in the treatment of CLL occurred during the study period (1995)(1996)(1997)(1998)(1999)(2000)(2001)(2002)(2003)(2004)(2005)(2006)(2007)(2008)(2009)(2010)(2011)(2012), with the introduction of monoclonal antibodies and the development of chemoimmunotherapy. As initial therapy, 153 (36%) patients received chemoimmunotherapy, 67 (16%) received a single-agent purine nucleoside analog, 60 (14%) received monoclonal antibody therapy with or without corticosteroids, 59 (14%) received single-agent alkylator therapy with or without corticosteroids, 42 (10%) received combination chemotherapy consisting of an alkylator with (n=24) or without (n=18) rituximab, 17 (4%) received a combination of purine nucleoside analog and alkylator, and 28 (7%) patients received other treatments. A higher proportion of patients in the group ≤55 years old received chemoimmunotherapy (36% versus 27%, P=0.005), single-agent purine nucleoside analog (16% versus 8%, P<0.001), or a combination of purine nucleoside analog and alkylator therapy (4% versus 2%, P=0.009) compared to patients >55 years. Fifty-five patients (7%) underwent an allogeneic stem cell transplant in the ≤55 years age group compared to 23 (1%) patients in the >55 age group (P<0.0001). The association between age and allogeneic stem cell transplant persisted when patients ≤55 years were further stratified by age with those ≤ 45 years (14%; 28/204) of age at diagnosis more likely to undergo stem cell transplantation than those 46-50 years (5%; 14/272) or 51-55 (4%; 13/ 368) years of age (P<0.0001). Time to first treatment and overall survival After a median follow-up of 5.5 years (range, 0-17 years), 1095/3168 (25%) patients have died; 192 (23%) in the ≤55 year old group and 903 (39%) in the >55 year old group. For the entire group, the median time to first treatment was 4.8 years, and the median overall survival was CLL in young patients haematologica | 2014; 99 Comparison of overall survival with the general population As a group, CLL patients ≤55 years old have significantly shorter overall survival than the age-and sex-matched population (median CLL=12.5 years; median population=not reached; P<0.0001, Among the 358 young patients with both FISH and IGHV results available, 171 were in Rai stage 0. Of these 171 patients, 97 (56.7%) had both mutated IGHV and a favorable FISH risk category, 54 (31.5%) had one unfavorable factor and 20 (11.7%) had two unfavorable factors. The median survivals in the groups with 0, 1, and 2 unfavorable factors were not reached, 13.0, and 7.7 years, respectively (P<0.001, Discussion In the past decade, considerable advances have been made in our ability to predict outcome and treat patients with CLL. This study is the first comprehensive report on the clinical characteristics and outcomes of a cohort of young CLL patients (≤55 years old) in the modern era and is approximately four times larger than any previous series of young CLL patients. Furthermore, information on IGHV gene mutation status and genetic abnormalities detected by FISH was available for slightly more than half (55%) of the patients, providing insight into the associations between age and molecular biomarkers. Significant differences in the presentation of young CLL patients relative to older patients were observed: young patients were more likely to present with intermediate Rai risk disease, have unmutated IGHV gene mutation status and express ZAP-70. Although the overall survival of young CLL patients as a group is longer than that of CLL patients >55 years old, the survival of all CLL patients is markedly shorter than that of their age-and sex-matched population. To the best of our knowledge, our study is the first to report that young CLL patients have a higher prevalence of adverse clinical and biological characteristics. These differences persisted when we limited our analysis to nonreferred patients and became even more pronounced when patients ≤55 years old underwent further age substratification. This observation suggests that young CLL patients are more likely than older patients to have biologically aggressive disease. CLL patients aged ≤55 years old were treated sooner than those >55 years old (median time to first treatment: 4 versus 5.2 years, respectively). It is possible that healthcare providers may have a bias towards treating young CLL patients sooner than older CLL patients who may have multiple other co-morbidities. However, the fact that the shorter time to first treatment among young patients was no longer significant after adjusting for Rai stage and IGHV mutation status suggests that the shorter time to first treatment among young patients is driven by differences in disease biology rather than age or the impact of co-morbidities. There were no differences noted in time to first treatment among the subgroups aged ≤45, 46-50 and 51-55 years old. In the 1990s, Montserrat et al. reported that the overall survival of young CLL patients is worse than that of the age-and sex-matched normal population. 9 More than 20 years later, our study confirms these findings. However, with the inclusion of several novel prognostic markers, we identified a subset of young patients whose survival is similar to that of the age-and sex-matched general population. Among the ~50% of the young patients in our series who had Rai stage 0 disease, slightly more than half (25% of all young patients) had both mutated IGHV and favorable FISH findings. The 15-year survival rate in these patients was >90%. Such individuals should be carefully followed to prevent disease-related complications -with attention to vaccinations, vitamin D levels, skin cancer surveillance, and monitoring for infectious and autoimmune complications of CLL. The rest of the patients are at high risk of disease progression and represent ideal candidates for enrollment into early intervention trials. This would allow us to study the impact of novel treatments in altering the natural history of this disease, and improve disease-free survival and overall survival. As expected based on transplant age-related eligibility considerations, young CLL patients were more likely to undergo stem cell transplantation. One in 16 patients ≤55 years old received a stem cell transplant. Notably, while age alone would not be an exclusion criterion to stem cell transplantation in any patient ≤55 years old, the proportion of patients who underwent stem cell transplantation became even more pronounced when patients aged ≤55 years old were sub-stratified further by age. In the group ≤45 years old, 14% received a stem cell transplant compared to 5% in the group 46-50 years old and 4% in the group 51-55 years old. The highest proportion of stem cell transplantation was seen in patients <40 years of age among whom one out of every five patients was eventually transplanted. How do these results compare to those of historical studies of young CLL patients? The influence of age on the outcome of patients with CLL was first described in the seminal publication by Rai et al. in 1975. 39 In this classic paper, 25 patients <50 years of age had a median overall S.A. Parikh et al. 144 haematologica | 2014; 99(1) 8 published their experience of 53 young CLL patients. There were no significant differences in traditional prognostic factors between these patients and older adults; the median overall survival was 7.1 years compared to 4.1 in the older age group. Two subsequent retrospective series (each with ~200 patients aged ≤55 years) confirmed these results. 7,30 Although the vast majority of the aforementioned studies did not report any differences in the traditional prognostic parameters between young and old CLL patients, we did find significant differences in these two populations of patients in the modern era. It is notable that cohorts of patients with CLL cared for in the 1970s and 1990s all had the same median overall survival of 12 years. This is exactly what we find in our study more than three decades later, which is a reminder that we need to be vigilant in developing and offering treatment options that improve not only disease-free survival but overall survival as well. Our study has several limitations. Not all patients had novel molecular/biological parameters available for inclusion in multiple analyses, given that these have only been used routinely in the past 5-10 years. Also, the treatment options for patients with CLL have undergone considerable changes during the study period. In addition, this was a single-center, retrospective analysis which needs validation among other cohorts of patients. Although we found that young CLL patients in Rai 0 stage with favorable IGHV and FISH results have a comparable survival to that of the general population over a 10-year interval, prolonged follow-up will be necessary to determine if this holds true in the long term. In summary, this study helps to formulate general concepts regarding the management and natural history of patients who are diagnosed with CLL well under the median age of presentation of this disease. Young CLL patients have an adverse clinical and molecular risk profile (i.e, Rai stage, IGHV mutation status) compared to older patients. As a collective group, the median overall survival of CLL patients aged ≤55 is 12 years -which is significantly shorter than that of the age-and sex-matched population. Despite this discouraging group average, we found that approximately 25% of young patients in our study who have Rai Stage 0 disease and either mutated IGHV genes or a favorable FISH risk category (such as normal cytogenetics or 13q-) have a survival comparable to that of the age-and sex-matched general population. Accordingly, information from prognostic testing of molecular biomarkers can be used to stratify outcome in younger patients and identify those most likely to benefit from participation in trials testing early intervention strategies. Leukemia and Lymphoma Society. Acknowledgment Dr. Shanafelt is a clinical scholar of th