SET alpha and SET beta mRNA isoforms in chronic lymphocytic leukaemia.

Alteration in RNA splicing is implicated in carcinogenesis and progression. Mutations in spliceosome genes and alternative splicing of other genes have been noted in chronic lymphocytic leukaemia (CLL), a common B cell malignancy with heterogeneous outcomes. We previously demonstrated that differences in the amount of SET oncoprotein (a physiological inhibitor of the serine/threonine phosphatase, PP2A) is associated with clinical aggressiveness in patients with CLL. It is unknown if alternative splicing of gene transcripts regulating kinases and phosphatases affects disease pathobiology and CLL progression. We show here for the first time that mRNA levels of the alternatively spliced SET isoforms, SETA and SETB (SETα and SETß), significantly correlate with disease severity (overall survival and time-to-first-treatment) in CLL patients. In addition, we demonstrate that relative increase of SETA to SETB mRNA can discriminate patients with a more aggressive disease course within the otherwise favourable CLL risk classifications of IGHV mutated and favourable hierarchical fluorescence in situ hybridisation groups. We validate our finding by showing comparable relationships of SET mRNA with disease outcomes using samples from an independent CLL cohort from a separate institution. These findings indicate that alternative splicing of SET, and potentially other signalling cascade molecules, influences CLL biology and patient outcomes.

Clinical outcomes in chronic lymphocytic leukaemia associated with expression of CD5, a negative regulator of B-cell receptor signalling.

Chronic lymphocytic leukaemia (CLL) is characterized by expression of CD5 on clonal B cells, and is partly driven by activated B-cell receptor (BCR) signalling. While CD5 is known to be a negative regulator of BCR signalling, it is unknown if variability in CD5 expression exists among patients and whether CLL cell CD5 expression affects CLL clinical outcomes. We assessed the extent to which CD5 expression is correlated with clinical outcomes, and whether this information adds to currently used prognostic markers. We evaluated CD5 expression from 1275 blood samples, established prognostic markers and time to event data from 423 CLL patients followed at the Duke University and Durham VA Medical Centers. CD5 median fluorescence intensity (MFI) was largely stable over time in individual patients, but ranged between 0·5 and 760 in the entire cohort. Lower CD5 MFI was significantly associated with a shorter time to first therapy. CD5 MFI, combined with established clinical and molecular prognostic markers, significantly improved risk-stratification. CD5 may affect disease outcomes by suppressing signalling through the BCR. Thus, a strategy to modulate CLL cell CD5 expression or function could be a therapeutic approach in CLL.

Relationship of blood monocytes with chronic lymphocytic leukemia aggressiveness and outcomes: a multi-institutional study.

Monocyte-derived cells, constituents of the cancer microenvironment, support chronic lymphocytic leukemia (CLL) cell survival in vitro via direct cell-cell interaction and secreted factors. We hypothesized that circulating absolute monocyte count (AMC) reflects the monocyte-derived cells in the microenvironment, and that higher AMC is associated with increased CLL cell survival in vivo and thus inferior CLL patient outcomes. We assessed the extent to which AMC at diagnosis of CLL is correlated with clinical outcomes, and whether this information adds to currently used prognostic markers. We evaluated AMC, clinically used prognostic markers, and time to event data from 1,168 CLL patients followed at the Mayo Clinic, the Duke University Medical Center, and the Durham VA Medical Center. Elevated AMC was significantly associated with inferior clinical outcomes, including time to first therapy (TTT) and overall survival (OS). AMC combined with established clinical and molecular prognostic markers significantly improved risk-stratification of CLL patients for TTT. As an elevated AMC at diagnosis is associated with accelerated disease progression, and monocyte-derived cells in the CLL microenvironment promote CLL cell survival and proliferation, these findings suggest that monocytes and monocyte-derived cells are rational therapeutic targets in CLL. Am. J. Hematol. 91:687-691, 2016. © 2016 Wiley Periodicals, Inc.

Veterans with blood cancers: Clinical trial navigation and the challenge of rurality.

PURPOSE: The proportion of cancer patients who participate in clinical trials (CTs) remains low, despite an understanding of barriers to enrollment. The barrier of rural residence is relevant to Veterans, who more commonly live in rural areas than non-Veterans. In this exploratory study, we aimed to examine geographic factors that could impede CT enrollment and to improve access to CTs for Veterans. METHODS: To assess the influence of rurality on the availability of CTs, we performed simulated searches using The Leukemia & Lymphoma Society's Clinical Trial Support Center (LLS CTSC) database. The LLS CTSC provides free CT education and navigation. In the second part of this study, we offered Veterans with blood cancers who received care at the Durham, Salem, Clarksburg, Sioux Falls, and Houston Veterans Administration (VA) Medical Centers referral to the LLS CTSC. FINDINGS: In simulated searches, we found significantly lower numbers of CTs open to enrollment in rural areas, compared to urban areas. In actual referrals, 33 Veterans were referred to the LLS CTSC, of which 15 (45%) lived in rural areas. Three Veterans enrolled in CTs. Patients declined referral or did not enroll in CTs for various reasons, including a desire to maintain care within the VA and/or to initiate therapy quickly. CONCLUSIONS: We identified "clinical trial deserts," which might hinder access and reduce CT participation for rural Veterans. Referral to the LLS CTSC promoted CT education and enrollment among a highly rural cohort of Veterans receiving care in the VA system.

SET oncoprotein overexpression in B-cell chronic lymphocytic leukemia and non-Hodgkin lymphoma: a predictor of aggressive disease and a new treatment target.

B-cell chronic lymphocytic leukemia (CLL), an incurable leukemia, is characterized by defective apoptosis. We found that the SET oncoprotein, a potent inhibitor of the protein phosphatase 2A (PP2A) tumor suppressor, is overexpressed in primary CLL cells and B-cell non-Hodgkin lymphoma (NHL) cell line cells. In CLL, increased levels of SET correlated significantly with disease severity (shorter time to treatment and overall survival). We developed SET antagonist peptides that bound SET, increased cellular PP2A activity, decreased Mcl-1 expression, and displayed selective cytotoxicity for CLL and NHL cells in vitro. In addition, shRNA for SET was cytotoxic for NHL cells in vitro. The SET antagonist peptide COG449 inhibited growth of NHL tumor xenografts in mice. These data demonstrate that SET is a new treatment target in B-cell malignancies and that SET antagonists represent novel agents for treatment of CLL and NHL.

Deep sequencing of the small RNA transcriptome of normal and malignant human B cells identifies hundreds of novel microRNAs.

A role for microRNA (miRNA) has been recognized in nearly every biologic system examined thus far. A complete delineation of their role must be preceded by the identification of all miRNAs present in any system. We elucidated the complete small RNA transcriptome of normal and malignant B cells through deep sequencing of 31 normal and malignant human B-cell samples that comprise the spectrum of B-cell differentiation and common malignant phenotypes. We identified the expression of 333 known miRNAs, which is more than twice the number previously recognized in any tissue type. We further identified the expression of 286 candidate novel miRNAs in normal and malignant B cells. These miRNAs were validated at a high rate (92%) using quantitative polymerase chain reaction, and we demonstrated their application in the distinction of clinically relevant subgroups of lymphoma. We further demonstrated that a novel miRNA cluster, previously annotated as a hypothetical gene LOC100130622, contains 6 novel miRNAs that regulate the transforming growth factor-ß pathway. Thus, our work suggests that more than a third of the miRNAs present in most cellular types are currently unknown and that these miRNAs may regulate important cellular functions.

Integration of Patient-Reported Outcome Measures in the Electronic Health Record: The Veterans Affairs Experience.

PURPOSE: There are growing efforts to integrate patient-reported outcome (PRO) data into electronic health records (EHRs) to bring together disparate sources of patient information and improve medical care. PRO measures can be used to assess cancer symptom presence and severity. Integrating PRO tools in EHRs can alert providers to address symptoms, which is an essential component of comprehensive oncology care. METHODS: We modified a PRO used to measure cancer and end-of-life symptoms, the Edmonton Symptom Assessment System to create the Veteran Symptom Assessment System (VSAS). VSAS was implemented as an integrated PRO as part of the Veterans Administration EHR system and was used at hematology-oncology clinics in Veteran Administration (VA) medical centers in the Southeast. RESULTS: From 2013 to 2014, VSAS was introduced, underwent usability testing and modifications, and was finally implemented in the EHR. Between 2015 and 2019, VSAS was administered 43,883 times in 9,058 patients. Eighty-nine percent of Veterans were male, 11% were female, 52% identified as non-Hispanic White, and 43% identified as African American. Fatigue, shortness of breath with exertion, and pain were most frequently reported initially (68%, 48%, and 45%, respectively) and were most frequently rated as severe (27%, 16%, and 17%, respectively). In patients diagnosed with stage IV cancer, higher symptom burden was significantly associated with shorter overall survival. The majority of Veterans with longitudinal measurements experienced improvement in symptoms, most frequently in severe symptoms. CONCLUSION: To our knowledge, this is the first large-scale implementation of a PRO system, integrated in the VA EHR, in ambulatory patients with cancer and blood disorders. The integration of VSAS within the VA EHR is a significant demonstration and a necessary requirement for current and future systemic initiatives in cancer symptom management.

Patterns of microRNA expression characterize stages of human B-cell differentiation.

Mature B-cell differentiation provides an important mechanism for the acquisition of adaptive immunity. Malignancies derived from mature B cells constitute the majority of leukemias and lymphomas. These malignancies often maintain the characteristics of the normal B cells that they are derived from, a feature that is frequently used in their diagnosis. The role of microRNAs in mature B cells is largely unknown. Through concomitant microRNA and mRNA profiling, we demonstrate a potential regulatory role for microRNAs at every stage of the mature B-cell differentiation process. In addition, we have experimentally identified a direct role for the microRNA regulation of key transcription factors in B-cell differentiation: LMO2 and PRDM1 (Blimp1). We also profiled the microRNA of B-cell tumors derived from diffuse large B-cell lymphoma, Burkitt lymphoma, and chronic lymphocytic leukemia. We found that, in contrast to many other malignancies, common B-cell malignancies do not down-regulate microRNA expression. Although these tumors could be distinguished from each other with use of microRNA expression, each tumor type maintained the expression of the lineage-specific microRNAs. Expression of these lineage-specific microRNAs could correctly predict the lineage of B-cell malignancies in more than 95% of the cases. Thus, our data demonstrate that microRNAs may be important in maintaining the mature B-cell phenotype in normal and malignant B cells.

Sequential Targeted Treatment for a Geriatric Patient with Acute Myeloid Leukemia with Concurrent FLT3-TKD and IDH1 Mutations.

Targeting and monitoring several acute myeloid leukemia mutations sequentially provides insights into optimal treatment plans.

Reflections on and future of hematologic malignancies research in the Veterans Health Administration.

Research in the Veterans Health Administration (VHA) has played an integral part in learning about cancer biology and treatment. Here we provide examples of past research performed in the VHA focusing on hematologic malignancies, and identify future opportunities for areas of research in this group of uncommon diseases that have specific importance for Veterans and the VHA. Veterans treated in the VHA and in the private sector deserve information that is focused on them, and is not an extrapolation from the larger population. Only by building upon and expanding existing research within the VHA can Veteran-specific results be collected and best practices be developed. In turn, such advances will benefit Veterans affected by these cancers with an improved quality of life and a longer lifespan.

Cocaine-induced pseudovasculitis.

Pseudovasculitis is a disease process that mimics the presentation and possibly the laboratory findings of true vasculitis. However, biopsy specimens do not reveal the typical histopathologic findings expected in vasculitis. One often overlooked cause of pseudovasculitis is cocaine use, which has been described in case reports to cause aggressive nasal destruction and various skin lesions and thus has been confused with Wegener granulomatosis or leukocytoclastic vasculitis. Unfortunately, serologic tests such as antinuclear antibody or antineutrophil cytoplasmic antibody cannot reliably differentiate between these entities. We describe a patient who presented with what was believed to be Wegener granulomatosis affecting the skin and upper airway. However, findings from repeated biopsies did not support this diagnosis, and the only unifying diagnosis was cocaine-induced pseudovasculitis. The ability to recognize and differentiate between true vasculitis and pseudovasculitis is essential for the clinician because treatment options are radically disparate.

Perifosine as a potential novel anti-telomerase therapy.

Most tumors circumvent telomere-length imposed replicative limits through expression of telomerase, the reverse transcriptase that maintains telomere length. Substantial evidence that AKT activity is required for telomerase activity exists, indicating that AKT inhibitors may also function as telomerase inhibitors. This possibility has not been investigated in a clinical context despite many clinical trials evaluating AKT inhibitors. We tested if Perifosine, an AKT inhibitor in clinical trials, inhibits telomerase activity and telomere maintenance in tissue culture and orthotopic xenograft models as well as in purified CLL samples from a phase II Perifosine clinical trial. We demonstrate that Perifosine inhibits telomerase activity and induces telomere shortening in a wide variety of cell lines in vitro, though there is substantial heterogeneity in long-term responses to Perifosine between cell lines. Perifosine did reduce primary breast cancer orthotopic xenograft tumor size, but did not impact metastatic burden in a statistically significant manner. However, Perifosine reduced telomerase activity in four of six CLL patients evaluated. Two of the patients were treated for four to six months and shortening of the shortest telomeres occurred in both patients' cells. These results indicate that it may be possible to repurpose Perifosine or other AKT pathway inhibitors as a novel approach to targeting telomerase.

Utilization of the Eµ-Myc mouse to model heterogeneity of therapeutic response.

Human aggressive B-cell non-Hodgkin lymphomas (NHL) encompass the continuum between Burkitt lymphoma and diffuse large B-cell lymphoma (DLBCL), and display considerable clinical and biologic heterogeneity, most notably related to therapy response. We previously showed that lymphomas arising in the Eµ-Myc transgenic mouse are heterogeneous, mirroring genomic differences between Burkitt lymphoma and DLBCL. Given clinical heterogeneity in NHL and the need to develop strategies to match therapeutics with discrete forms of disease, we investigated the extent to which genomic variation in the Eµ-Myc model predicts response to therapy. We used genomic analyses to classify Eµ-Myc lymphomas, link Eµ-Myc lymphomas with NHL subtypes, and identify lymphomas with predicted resistance to conventional and NF-κB-targeted therapies. Experimental evaluation of these predictions links genomic profiles with distinct outcomes to conventional and targeted therapies in the Eµ-Myc model, and establishes a framework to test novel targeted therapies or combination therapies in specific genomically defined lymphoma subgroups. In turn, this will rationally inform the design of new treatment options for aggressive human NHL.

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.

Clinical and biological relevance of genomic heterogeneity in chronic lymphocytic leukemia.

BACKGROUND: Chronic lymphocytic leukemia (CLL) is typically regarded as an indolent B-cell malignancy. However, there is wide variability with regards to need for therapy, time to progressive disease, and treatment response. This clinical variability is due, in part, to biological heterogeneity between individual patients' leukemias. While much has been learned about this biological variation using genomic approaches, it is unclear whether such efforts have sufficiently evaluated biological and clinical heterogeneity in CLL. METHODS: To study the extent of genomic variability in CLL and the biological and clinical attributes of genomic classification in CLL, we evaluated 893 unique CLL samples from fifteen publicly available gene expression profiling datasets. We used unsupervised approaches to divide the data into subgroups, evaluated the biological pathways and genetic aberrations that were associated with the subgroups, and compared prognostic and clinical outcome data between the subgroups. RESULTS: Using an unsupervised approach, we determined that approximately 600 CLL samples are needed to define the spectrum of diversity in CLL genomic expression. We identified seven genomically-defined CLL subgroups that have distinct biological properties, are associated with specific chromosomal deletions and amplifications, and have marked differences in molecular prognostic markers and clinical outcomes. CONCLUSIONS: Our results indicate that investigations focusing on small numbers of patient samples likely provide a biased outlook on CLL biology. These findings may have important implications in identifying patients who should be treated with specific targeted therapies, which could have efficacy against CLL cells that rely on specific biological pathways.

Phase II study of cenersen, an antisense inhibitor of p53, in combination with fludarabine, cyclophosphamide and rituximab for high-risk chronic lymphocytic leukemia.

Patients with chronic lymphocytic leukemia (CLL) with deletion or mutation of TP53 have exceedingly poor clinical outcomes. Cenersen, an oligonucleotide targeting TP53, has been shown to abrogate the activity of TP53 gain-of-function mutants and to increase sensitivity of lymphoma cells to cytotoxic chemotherapy in vitro. We combined cenersen with fludarabine, cyclophosphamide and rituximab (FCR) as treatment for patients with high-risk CLL. The purpose of this phase II study was to determine the overall response rate, response duration and toxicity of cenersen administered in combination with FCR. Twenty patients with relapsed or high-risk CLL were evaluated. Nineteen patients were previously treated. The complete response rate was 18%; the overall response rate was 53%. Median progression-free and overall survival was 5.3 and 10.6 months, respectively. The most common serious adverse events were neutropenia and thrombocytopenia. In this single arm phase II study, cenersen combined with FCR yielded clinical responses with acceptable toxicity in patients with high-risk CLL.