ALBERT

Description: This phase 1 clinical trial evaluated the safety and effects of autologous dendritic cells (DC) injected to patients in late chronic or accelerated phase of chronic myelogenous leukemia (CML). Seven patients with a median age of 63 years (range 55–78) entered the trial. Median time from diagnosis to registration was 38 months (range 16–94). Two patients were in the accelerated phase of disease based on the presence of additional bone marrow chromosomal abnormalities, all other patients were in chronic phase. Prior therapies included hydroxyurea and alpha interferon with or without cytosine arabinoside. Four patients were on therapy with imatinib mesylate during the course of the trial, two were treated before imatinib became available and one was previously intolerant of imatinib. CD14+ monocytes were selected from a leukapheresis product by clinical grade immunoadsorption (Miltenyi Biotec). The monocytes were matured to CD83+ and bcr-abl+ DC after 10 days in culture with X-VIVO 15 medium with 1% human AB serum, GM-CSF, IL-4, as the base culture media followed by maturation mediated by TNF-alpha, IL-6 and prostaglandin E2. The first patient entered had a positive mycoplasma reaction by polymerase chain reaction at the time of his first DC injection, the cells were not given and he was withdrawn from the study. The other six patients were treated in two cohorts of three patients and received four injections each of 3x106 DC and 15x106 DC per injection, respectively. CML-DC injections were generally well tolerated and all non-hematologic toxicities were ≤ grade 2. All injections were given intra-nodally into a cervical lymph node except in the first patient where the DCs were injected subcutaneously. We observed no major clinical responses, but the white blood cell counts normalized or were maintained at normal level in four patients during therapy. Three patients experienced a transient reduction in the percentage of bcr-abl+ cells suggesting that the treatment may have affected the cells in a leukemia specific manner. We detected vascular endothelial growth factor (VEGF) in the plasma prior to therapy. In four of five patients, VEGF levels decreased by more than 75 percent during therapy. T cells isolated from patients’ blood and incubated with autologous CML DC before, during and after therapy showed increased proliferation and secretion of interferon gamma during the course of therapy. DCs from three patients were transfected with a recombinant replication-defective adenovirus carrying the gene for IL-2 under a constitutively active promoter. IL-2 secreted by transfected dendritic cells enhanced in vitro T cell proliferation and interferon gamma release and these effects increased with increasing numbers of DC injections. This study demonstrates that CML-DC can be prepared from the blood of patients treated with imatinib mesylate, that they can be administered safely and that progress of therapy is accompanied by an increase in DC-specific T cell reactivity and a decrease in circulating VEGF.

Description: Abstract 2807 CCDC26 is a retinoic acid-dependent modulator of myeloid cell differentiation and death (Yin W et al. Oncogene 2006 :25 :3735). CCDC26 maps to chromosome 8q24.21 and germline polymorphisms in the gene have been associated with low-grade gliomas (Jenkins RB et al. Cancer Genetics 2011;204 :13), which in turn have recently been shown to frequently express IDH1 mutations (Parsons D et al, Science 2008;321 :1807). More recently, we have discovered that CCDC26 SNP associations in gliomas are primarily restricted to patients with IDH1 mutations (Jenkins et al. unpublished observations). We therefore hypothesized that germline CCDC26 SNPS might be associated with patients with myeloid malignancies that harbor IDH1 mutations (Mardis et al. NEJM 2009;361 :1058). Seventy-two IDH -mutated patients with chronic or blast phase myeloproliferative neoplasms (MPN), myelodysplastic syndromes or acute myeloid leukemia (most constituting post-MPN events) were identified: 18 IDH1 R132, one IDH2 R172 and 53 IDH2 R140. These 72 patients and a control group of 153 IDH unmutated cases with myeloproliferative or myelodysplastic neoplasms (total n =225) were genotyped for CCDC26 risk alleles associated with IDH -mutated gliomas: rs10464870, rs891835 and rs4295627. The respective minor allele frequencies (MAF) were significantly different between IDH1 (11%, 8% and 6%) and IDH2 (30%, 27% and 24%) mutated cases (p=0.037, 0.040 and 0.021; OR 3.4, 3.7 and 6.1 with 95% CI of 1.1–10.5, 1.1–13.0 and 1.3–28.9) as well as between IDH- wild type (24%, 24% and 21%) and IDH1 -mutated (11%, 8% and 6%) cases (p=0.10, 0.043 and 0.035; OR 0.42, 0.29 and 0.21 with 95% CI of 0.15–1.19, 0.09–0.96 and 0.05–0.90). The MAF values in our IDH -unmutated cases were similar to that of population-based controls. However, the direction of SNP associations in IDH1 -mutated myeloid malignancies were opposite of those seen in IDH1 -mutated gliomas. Our observations suggest a differential effect of CCDC26 genetic polymorphisms in IDH1 -mutated myeloid malignancies compared to either their IDH2 R140-mutated or IDH1 -mutated gliomas. Such differences in genomic context might contribute to hitherto seen differences in prognostic impact between IDH2 R140 vs. IDH1 R132 vs. IDH2 R172, in AML (Green CL et al. Blood Published online before print May 19, 2011, doi: 10.1182/blood-2010-12-322479). Disclosures: No relevant conflicts of interest to declare.

Description: Peripheral T- and NK-cell lymphomas (PTCLs) are fatal in the majority of patients, and conventional chemotherapy is minimally effective. Targeted inhibition of tyrosine kinases (TKs) might be a useful treatment strategy, but TK overexpression by PTCLs has not been well characterized (except ALK). Recently a novel translocation t(5;9) fusing the ITK and SYK genes was reported in a subset of unspecified-type PTCLs, associated with high SYK gene expression and Syk protein positivity. Syk is a non-receptor TK important in immunoreceptor signal transduction, providing proliferation and survival signals in a variety of hematopoietic cells. Syk is absent or expressed at very low levels in most normal post-thymic T cells, where this signal transduction role is fulfilled by ZAP70. Since Syk TK inhibitors are now in clinical trials for other diseases, we sought to determine the incidence of t(5;9) and Syk overexpression in PTCL. Tissue microarrays were constructed using triplicate cores from paraffin-embedded tissue blocks. Cases were studied using fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC). Additional studies were performed using flow cytometry and Western blot. There were 141 PTCLs, including 35 angioimmunoblastic, 66 unspecified, 21 anaplastic large cell, 2 enteropathy-associated, 5 hepatosplenic, 5 extranodal NK/T, and 7 other. No evidence of t(5;9) was identified by FISH in 86 informative cases. Normal T-cell subsets from peripheral blood, spleen, and lymph node were negative for Syk by IHC, flow cytometry, and Western blot. Syk was positive by immunohistochemistry in 133 of 141 (94%) PTCLs. All 8 Syk negative cases were extranodal, cytotoxic PTCLs. Western blot on frozen tissue correlated with IHC results in paraffin. All 4 Syk-positive cases tested showed phosphorylation of tyrosine residues 525/526, known to be required for Syk activation. We conclude that Syk is overexpressed in most PTCLs, including the vast majority of nodal cases. Syk may substitute for ZAP70 in these tumors and provide ongoing proliferation and survival signaling. The overexpression of Syk and phosphorylation of its Y525/526 residues suggest Syk may be a suitable target for pharmacologic TK inhibition as a treatment option for patients with PTCL.

Description: Genetic analysis has an increasing role in the diagnosis, classification, and management of patients with CLL. Although translocations involving the IgH locus are well characterized in B-cell malignancies such as follicular cell lymphoma, their frequency and significance in CLL are less well defined. We studied a series of patients with a clinical diagnosis of CLL using a FISH probe set including IgH. Methods: FISH was performed on blood using a DNA probe set designed to detect common chromosome anomalies associated with CLL and translocations involving IgH. Patients with an IgH abnormality were further studied using FISH probes sets for IgH and cyclin D1, BCL-2, BCL-3, BCL-6, BCL-11a, c-MYC, MALT-1, c-MAF, FGFR-3 or PAX-5. The clinical presentation, pathology, and flow cytometric immunophenotype was reviewed in patients with various translocations involving IgH. Results: Between 1/1/03 and 6/15/04, samples from 1032 patients with a clinical diagnosis of CLL were analyzed by FISH. Of these, 76 patients (7%) had translocations involving the IgH locus. Sixty one patients had a single translocation partner gene (cyclin D1 45%, BCL-2 24%, BCL-3 8%, c-MYC 2.5%, or BCL-11a 1.3%). Two patients had two independent clones (cyclin D1 and BCL-2; BCL-3 and BCL-11a). In 13 pts (17%) we could not identify the translocation partner. Thirty five (46%) of the 76 patients with IgH translocations had been seen at Mayo Clinic Rochester and had clinical and laboratory data for detailed review. Among these 35 patients, 16 (45%) had fusion of IgH and cyclin D1 and were diagnosed with mantle cell lymphoma (MCL). The immunophenotype of the monoclonal B cells was typical of MCL (CD5+, CD23-) in 6 patients and typical of CLL (CD5+, CD23+) in 4 patients. Six MCL patients had non-diagnostic immunophenotypes: CD5-, CD 23+ (n = 3) and CD5-, CD23- (n = 3). Twelve (34%) of the 35 patients had the IGH/BCL-2 fusion gene. Of these, 3 patients had leukemic phase follicular lymphoma, one had both CLL and MCL, and 8 had the diagnostic immunophenotype of CLL. The remaining 7 patients (19%) had a variety of translocation partners: BCL-3 (n = 1), BCL-3 and BCL-11a (n = 1), c-MYC (n = 2), and unknown (n = 3). The patients with a translocation involving BCL-3 had a typical CLL immunophenotype. Discussion: The IgH FISH probe detected a molecular defect in 7% of patients with a circulating monoclonal B cell population. In patients with a CLL immunophenotype, use of the IgH probe identified the IgH/cyclin D1 translocation characteristic of MCL in 4 patients, a finding of major clinical significance. Interestingly, after exclusion of MCL patients, more than 4% patients with CLL had other translocations involving IgH, a proportion comparable with other better defined chromosomal abnormalities in CLL. Although IgH/BCL-2 translocation is usually associated with follicular lymphoma, we identified 8 patients with CLL and this translocation. Translocations involving BCL-3 had been described before in CLL with an “atypical” immunophenotype. In contrast both of our patients with BCL-3 had typical phenotypes. Translocations involving IgH and BCL-11a have not been previously described. We conclude that FISH studies using the IgH probe improve the precision of diagnosis in CLL and could also have prognostic value.

Description: Introduction: A new isodicentric variant of del(20q) was recently described; namely ider(20)(p11.2)del(20)(q11.2q13.3). The frequency and clinical significance of this anomaly are not yet established. This anomaly is usually missed in routine cytogenetic studies and the common commercial FISH probe, D20S108, does not distinguish between del(20q) and ider(20q). The goals of this study were to determine 1) the frequency and hematopathology of patients (pts) with ider(20q) and 2) the best cytogenetic methods to distinguish among various del(20q) anomalies. Methods: We selected 12 pts referred for chromosome studies with -20,+mar to look for evidence of ider(20q). For comparison, we also randomly selected 12 pts with classical del(20q). Up to 10 bone marrow metaphases were studied for each of these 24 pts using FISH with probes for 20pter, JAG1 (20p12), centromere 20, D20S108 (20q12) and 20qter. We also studied 200 interphase nuclei from each pt using D20S108 and 20qter probes. We reviewed cytogenetic results for a consecutive series of pts in our hematological practice between 9/1/05 and 12/31/05 to establish the frequency of ider(20q). Blood and marrow slides were available for microscopic evaluation in 9 of 12 pts. with ider(20q). Results: By metaphase FISH, each of the 12 pts with del(20q) were 20pter+, JAG1+, D20S108- and 20qter+ indicating an interstitial deletion. Each of the 12 pts with ider(20q) were 20pter-, JAG1-, D20S108-- and 20qter++ indicating an isodicentric chromosome with an interstitial del(20q) on either side of the centromere. The ider(20q) is best detected using probe for D20S108 and 20qter. By FISH each pt with ider(20q) had a mixture of cells with del(20q) and ider(20q), though cells with only del(20q) were not usually detected by chromosome studies. One pt had a subclone with two copies of ider(20q). Interphase FISH identified del(20q) and ider(20q) in each specimen. Hematopathology studies of 9 pts with ider(20q) indicated acute myeloid leukemia (AML) with multilineage dysplasia in 1 and myelodysplastic syndrome in 8 (refractory cytopenia with multilineage dysplasia (RCMD) in 3, RCMD with ringed sideroblasts in 3, atypical myelodysplastic/myeloproliferative disease in 1, and refractory anemia with excess blasts-2 (RAEB-2) in 1). Additional chromosome anomalies were detected in the pts with AML with multilineage dysplasia and RAEB-2. Six pts had dysgranulopoiesis in addition to dysmegakaryopoiesis and/or dyserythropoiesis. Among 5,744 pts in our hematological clinical practice screened by interphase FISH, ider(20q) was seen in 8, but 8 of 78 pts with del(20q) also had ider(20q). Discussion: In clinical practice ider(20q) is common in hematological disorders and occurs in 0.14% of all pts and in 10% of pts with del(20q). This figure may underestimate the true frequency of ider(20q) because novel FISH studies with D20S108 and 20qter are required to confirm its presence. When G-banding preparations indicate -20,+mar, then ider(20q) should be suspected. The literature is sparse but pts with ider(20q) appeared to have a more consistent presentation of multilineage dysplasia with additional involvement of the granulocytic series than pts with del(20q). These results suggest that it is helpful to distinguish among various forms of del(20q) in clinical practice using both chromosome and FISH studies with D20S108 and 20qter.