ALBERT

Description: Flavopiridol has in vitro activity in CLL and promotes apoptosis independent of p53 function or prior fludarabine exposure. We sought to determine if flavopiridol administered using two different schedules has activity in CLL. Patients with previously treated CLL were enrolled on two sequentially performed phase II studies. Patients in the first trial received flavopiridol (50 mg/m2 daily) as a continuous infusion (CI) over 72-hours every 2 weeks. Patients in the second trial received flavopiridol 50 mg/m2 as a 1-hour intravenous bolus (IVB) daily for three days repeated every 3 weeks. Patients received up to 6 (CI cohort) or 8 (IVB cohort) cycles of therapy. Fifteen patients enrolled in the 72-hour CI phase II trial; 6 (40%) had intermediate (Rai stage I or II) and 9 (60%) high (Rai stage III and IV) risk stages. No responses were noted in this group with 27% having stable disease (SD) and 73% progressive disease (PD). Thirty-six patients enrolled in the IVB study, with 13 (36%) having intermediate and 23 (64%) having high-risk disease. Four patients (11%) had partial responses, 19 (53%) SD, and 13 (36%) PD. The progression-free survivals for responders in the IVB study were 2.9, 3.2, 8.7, and 19.3 months. The median progression-free survival was 2.1 months (95% confidence interval [CI] 1.8 – 3.8) for patients in the CI study and 3.2 months (95% CI [2.5 – 7.4]) for the IVB study. The median overall survival was 27 months (95% CI [20–42]) for the CI study and 24 months (95% CI [18–31]) for the IVB study. Toxicity was manageable and included mainly myelosuppression (granulocytopenia and thrombocytopenia), infections, diarrhea and fatigue. Grade 3 and 4 toxicities were 20% and 27%, respectively, on the CI study and 39% and 33% on the IVB study. One patient on the IVB study had tumor lysis syndrome that was managed medically and did not require dialysis. There was one on-study death following a myocardial infarction on the IVB study. We conclude that flavopiridol has modest, schedule-dependent clinical activity in relapsed CLL and warrants future investigation utilizing alternative schedules of administration.

Description: Cytogenetics and comparative genomic hybridization (CGH) have been used to identify large genomic amplifications and deletions in all subtypes of acute myeloid leukemia (AML). Up to 15–20% of AML patients have a normal karyotype at diagnosis. While cytogenetic abnormalities confer important prognostic information for patients with AML, there remain differences in the therapeutic response and outcome among patients with the same cytogenetic profile, implying that other, more subtle, genetic abnormalities may exist. We hypothesized that a subset of AML patients with normal cytogenetics may contain genomic DNA copy number changes that are too small to be detected using standard cytogenetic techniques. To address this possibility, we used high-resolution bacterial artificial chromosome (BAC) array CGH technology to examine 31 AML patients with normal cytogenetics. The BAC arrays contain 2,464 BAC clones spotted in triplicate on glass slides, and provide a 1 Mb resolution of the entire human genome. Technical generation of the arrays, hybridization parameters, and analysis were similar to that reported for murine BAC array CGH (Nat Genet. 2001 Dec;29(4):459–64). The 31 AML samples included 4 M0, 8 M1, 10 M2, and 9 M4 patients. Array CGH experiments were performed using 500 nanograms of Cyanine 5 labeled genomic DNA from unmanipulated AML bone marrow, mixed with an equal amount of control DNA (a pool of DNA from 4 cancer-free individuals) labeled with Cyanine 3. Using the human 1 Mb BAC arrays, we identified amplifications and deletions from multiple samples that were confirmed with G-banding cytogenetics [del(7)(q31), del(7)(p11.2), +8, del(11)(q13q23), +21, add(21)(q22), −X, −Y, +Y]. In addition, BAC arrays robustly detected copy number alterations that were identified in as few as 4/21 metaphases. We identified 5/31 (16%) patients with normal cytogenetics that contained altered genomic DNA copy numbers using BAC array CGH. Copy number changes were confirmed for several of these genomic loci using a dye-swap experiment, where the AML DNA was labeled with Cyanine 3, and the control DNA with Cyanine 5. Two of the 5 patients with abnormalities detected using array CGH would be reclassified from “intermediate” to “unfavorable” cytogenetics [del(7)(q31.31q34), add(11)(q23.3qter), and 17(p12pter)]. These results suggest that a subset of AML patients with normal cytogenetics contain genomic copy number alterations that may effect treatment and outcome. Patient # FAB subtype Genomic location Gain or loss Size (Megabase) Dye-Swap confirmed 1 M0 7(q31.31q34) loss 2.0 Not done 1 11(q23.3qter) gain 16.5 Not done 2 M1 11(p14) loss 7.4 Yes 3 M1 11(q13.2q14.1) gain 15.8 Yes 3 19(p) gain 64.0 Yes 4 M2 17(p12pter) gain 8.6 Not Done 5 M2 19(p13.1pter) loss 14.8 Yes 5 12(q13) loss 5.0 Yes

Description: Background: Fanconi anemia (FA) is caused by mutations in one of seventeen genes that make up the FA DNA double strand break (DSB) repair pathway. Recently, two individuals with biallelic germline BRCA1 mutations, each consisting of one null and one hypomorphic mutation, were identified and noted to have features consistent with FA, including congenital anomalies and increased chromosomal breakage of lymphocytes on exposure to diepoxybutane (Domchek et al. Cancer Discov. 2013 Apr; (4):399-405; and Sawyer et al. Cancer Discov. Epub. 2014 Dec 3.), adding BRCA1 as the newest FA gene. However, neither patient developed bone marrow failure (BMF), making the bone marrow effects of BRCA1 deficiency still unclear. Methods: To test the hypothesis that Brca1 is also essential in hematopoiesis, we developed a conditional mouse model with Mx-1 Cre-mediated Brca1 deletion and examined the effects of Brca1 deficiency on hematopoiesis in this model. Results: At baseline, Brca1-/- mice have macrocytic anemia and leukopenia. Further, by 6 months of age, 30% and 50% of the Brca1-/- mice develop spontaneous BMF or hematologic malignancies (HM), respectively. Brca1-/- mice develop a diverse range of HM, including T-cell lymphomas and acute myeloid leukemias, suggesting a defect in an early hematopoietic progenitor population. Methylcellulose colony forming assays also demonstrate a defect in progenitor cell function with Brca1-/- bone marrow cells forming fewer colonies (44.4±31.9) than Brca1+/+ cells (200.3±30.5, p=0.004) at baseline, and show FA-like hypersensitivity to the DNA cross-linking agent, Mitomycin C (MMC) (mean colony survival % at 10 nM MMC 40% versus 82% and at 50 nM 1% vs 56%). Spectral karyotyping of bone marrow cells from mice that developed BMF demonstrated chromatid exchanges and breaks. Similarly, multiple chromosomal translocations were seen in the myeloid leukemia cells, implicating genomic instability in the pathogenesis of these disorders. Conclusions: Taken together, our results show that loss of Brca1 in murine bone marrow causes hematopoietic defects and MMC sensitivity similar to that seen in humans with FA, providing strong evidence that Brca1 is critical for normal hematopoiesis and that Brca1 is a bona fide FA gene. This novel mouse model provides the opportunity to gain functional insight into the key stage(s) of hematopoiesis that require Brca1 and the effects of Brca1 haploinsufficiency, as seen in humans, on hematopoiesis. Further, as nearly all of the single gene FA mouse models to-date have failed to recapitulate the bone marrow phenotype of human FA, this model will be critical for deepening our understanding of the pathogenesis of FA. Disclosures No relevant conflicts of interest to declare.

Description: Disease- or treatment-associated chromatin conformation has yet to be illustrated. Here we first demonstrate that monocytic and erythroid leukemia cell lines have distinctly different chromatin conformation at the PU.1 locus: a looped, connected, RNA-polymerase-II-bound, active conformation in the former and a disconnected, inactive conformation in the latter. These conformations undergo opposite transformations, becoming more active in the erythroid leukemia line and less active in the monocytic leukemia line, in response to both DNA and histone hypomethylating drugs.To explore the underlying mechanisms we developed a novel method to analyze DNA modifications. We demonstrate that the erythroid leukemia line has a marked drug-responsive change in both hydroxymethyl-CpG and 5-meythyl-CpG at the promoter, whereas the monocytic leukemia line has a higher level bivalent histone/chromatin with co-localized H3K4me3 and H3K27me3 at the enhancer. Consequently, the erythroid leukemia line is more sensitive to DNA hypomethylation while the monocytic leukemia line is more sensitive to histone hypomethylation. Further studies on clinical leukemia samples confirm the findings, and demonstrate that the leukemic cells from the clinical samples have different chromatin conformations with much more bivalent chromatin and denser DNA/histone modifiers and are more sensitive to hypomethylating drugs, compared to normal controls. Different chromatin conformations dictate preferential drug responses and open new diagnostic and therapeutic avenues. Disclosures: No relevant conflicts of interest to declare.

Description: Peripheral T-cell lymphomas are uncommon and account for 10-15% of all non-Hodgkin lymphomas (NHL). The current classification and treatment strategy of peripheral T-cell lymphomas relies on integrating morphology with immunophenotype, genetics and clinical presentation. However, the most common category of peripheral T-cell lymphomas remains peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS) reflecting the lack of specific parameters to better define these lymphomas in a biologically relevant way. As our understanding of the biology of peripheral T-cell development continues to improve, several immunophenotypic markers have become available that can delineate peripheral T-cells into functional subsets. It is now recognized that peripheral T-cell lymphomas can arise from both conventional and innate-like T-cells. Classically, peripheral lymphomas with the γδ T-cell receptor (TCR) as well as lymphomas derived from true natural killer (NK) cells are considered to be arising from the innate T-cells whereas T-cell lymphomas with an αβ TCR are assumed to be derived from the adaptive, conventional T-cells. However, several recent studies have identified relatively rare populations of αβ T cells with extremely limited α chain diversity. These T-cells are characterized by the ability to mount immune responses by interacting with non-classical MHC class I antigen presenting molecules even in the absence of intentional priming. The cells within these populations express markers characteristic of NK cells and/or memory T-cells and include cells frequently labeled as “NKT” cells and mucosal-associated invariant T-cells (MAIT). Whether these cells contribute to peripheral T-cell lymphomas is not known. The transcription factor promyelocytic leukemia zinc finger (PLZF) is indispensable for development and maturation of these T-cells. We therefore asked the question whether PLZF expression could be used to identify peripheral T-cell lymphomas derived from these innate like non-conventional T-cells. To answer this question, we generated a tissue microarray that included biopsies from 26 PTCL-NOS, 11 anaplastic large cell lymphomas (ALCL), ALK-, and 13 ALCL, ALK+. Histologically normal tonsil, lymph node, thymus and gastrointestinal biopsies were used as controls. Immunohistochemistry with the PLZF antibody was performed in the clinical immunohistochemistry laboratory. Only rare PLZF positive cells were observed in uninvolved tonsils, lymph nodes, and thymus. In contrast, the intestinal mucosa, which is normally enriched in PLZF positive innate type T-cells showed a relative abundance with expression observed in 8-10% lymphocytes. Lymphomas were scored as positive when 20% or more of tumor cells showed expression with nuclear localization. Within the lymphomas, PLZF expression was observed in 2/26 PTCL-NOS and 2/13 ALCL, ALK+. PLZF expression was not observed in any of the ALCL, ALK- lymphomas included in the current study. PCR amplification followed by sequencing identified the Vα7.2-Jα33 TCR rearrangement characteristic of the MAIT cells in the two PLZF positive PTCL-NOS lymphomas confirming the origin of these lymphomas from bona fide innate-like T-cells. Sequencing of the TCR in the remaining three PLZF positive lymphomas is currently in progress. Cytogenetic analysis was available in three of the 4 cases. While t(2;5) was the sole cytogenetic abnormality in one ALCL, ALK+ lymphoma, the remaining two cases, including one ALCL, ALK+ had a complex karyotype that included t(2;5). In view of the relatively small number of patients available for analysis and the heterogeneous therapy administered to patients included retrospectively in the study, an outcome analysis was not performed. In conclusion, we demonstrate that PLZF expression identifies lymphomas derived from non-conventional innate-like T-cells and likely represent a biologically unique group of peripheral T-cell lymphoma. It is well known that innate T-cells are highly resistant to xenobiotics due to high expression of the transporter ATP binding cassette B1 (MDR). Prospective evaluation for PLZF expression may therefore be useful in identifying patients who will benefit from therapy that specifically targets this pathway of drug resistance. Disclosures: No relevant conflicts of interest to declare.

Description: Normal hematopoiesis is controlled by a well-connected genetic network composed of several transcription factors (TFs) including PU.1 and GATA1. It has been postulated that both transcription factors and epigenetic modifiers work collaboratively to regulate hematopoietic stem cell differentiation and lineage specification as well as leukemogenesis. However, it is unclear about how the interplay between genetic network and epigenetic regulatory modifiers regulates locus-specific chromatin modifications and gene expression in normal hematopoiesis and hematologic malignancies such as myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Drugs targeting epigenetic modifiers including DNA methyltransferases (DNMTs), histone methyltransferases (HMTs) and histone deacetylases (HDACs) have been shown to be effective in a small portion of patients with MDS/AML, but the mechanisms underlying the efficacy and selectivity of different epigenetic modifying drugs are unknown. In this study, we performed growth-inhibition experiments with several epigenetic modifying drugs in multiple AML cell lines and identified two distinct lineage/differentiation-associated growth-inhibition patterns. Monocytic leukemia cells, but not erythroid leukemia cells, were sensitive to H3K4 HMT inhibitors, whereas both erythroid and monocytic leukemia cells were hypersensitive to DNMT and H3K27 HMT inhibitors. Importantly, co-immunoprecipitation experiments demonstrated lineage-specific interactions between the lineage-determining TFs (PU.1/SPI1 and GATA1) and the DNA/histone modifiers (DNMT1, DNMT3A/3B, TET2 and EZH2). Specifically, SPI1/PU.1 interacts with DNMT1 and EZH2, while GATA1 interacts with TET2 and DNMT3A/3B in MDS-derived erythroid leukaemia. In monocytic leukemia, SPI1/PU.1 interacts with TET2. Epigenetic modifying drugs such as azacytidine and 3-deazaneplanocin efficiently disrupted the interactions between the lineage-determining TFs and the DNA/histone modifiers without changing the expression of these proteins. We developed a new method, crosslink-assisted DNA modification immunoprecipitation assay (CDMIA), to simultaneously measure 5-methylcytosine (5-mC) and hydroxymethylcytosine (5-hmC). CDMIAs revealed significant drug-responsive changes in 5-mC/5-hmC at the promoters of differentiation/lineage-controlling genes such as PU.1/SPI1, but not at the global 5-mC/5-hmC. Sequential-ChIP and chromatin conformation capture (3C) showed that PU.1/SPI1 recruited polymerase II (pol-II) and the DNA/histone modifying complexes to PU.1/SPI1 toform distinct chromatin structures in a lineage-specific manner. We have selected azacytidine-resistant clones and established azacytidine-resistant cell lines from the previously azacytine-sensitive erythroid and monocytic leukemia cells. Strikingly, azacytine at the same concentrations failed to disrupt the interactions between the lineage-determining transcription factors and the DNA/histone modifiers in these drug-resistant leukemia cells. Genome-wide sequencing revealed novel mutations in TET2, TET3, DNMT3L and PU.1/SP1 that were confirmed by Sanger sequencing. These mutations correlated with the altered interactions between PU.1/SPI1 and the DNA/histone modifying complexes and predicted the responses to epigenetic modifying drugs. Examination of clinical specimens from patients with MDS/AML confirmed the presence of distinct lineage/differentiation-specific chromatin structure with a high-level recruitment of DNA/histone modifiers. Our genome-wide epigenetic analysis demonstrates the statistically significant enrichment of the SPI1/PU.1, TP53 and MYB DNA-binding motifs in hyper-H3K27 trimethylated DNA sequences in erythroid-predominant MDS. These results demonstrate the presence of locus-specific, drug-sensitive chromatin structures in MDS/AML subtypes. Our data revealed a novel epigenetic modifying drug action model that involves selective disruption of the disease-specific interactions between the lineage-determining factors and DNA/histone modifiers. Such drug action models may provide new insights into the mechanisms underlying the efficacy and selectivity of epigenetic modifying drugs. Disclosures Larson: Novartis: Consultancy, Research Funding; Pfizer: Consultancy; Ariad: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy.

Description: In order to assess whether Pgp modulation with PSC833 improves disease-free (DFS) and overall (OS) survival in untreated patients (pts) with AML 〈 60 years, a phase 3 trial (CALGB 19808) was undertaken comparing two induction regimens: Ara-C (A), Daunorubicin (D), and Etoposide (E), with (ADEP) or without (ADE) PSC833. All pts received A 100 mg/m2 by CIV daily for 7 days. In ADE, D 90 mg/m2 and E 100 mg/m2 were each given daily by short IV infusion on days 1–3; the corresponding doses in ADEP were D 40 mg/m2 and E 40 mg/m2. PSC833 10 mg/m2 was given by CIV over 72 hrs after a loading dose of 2.8 mg/kg IV over 2 hrs. These doses were based on findings from parallel phase I trials that showed comparable efficacy and toxicity for the 2 regimens (JCO2004; 22:4290). Pts in complete remission (CR) received consolidation therapy according to cytogenetic risk (ASH2001, 688a) and were then randomized to a phase 3 immunotherapy sequence comparing interleukin-2 with observation. The Pgp modulation portion of the trial ended when PSC833 became unavailable after 302 of a planned 600 pts had been randomized. Prior myelodysplasia or therapy-related AML were exclusion criteria. The median age was 45 years; the arms were comparable with respect to cytogenetic risk groups (Core-Binding Factor (CBF) leukemia vs. others). Response by intent-to-treat on the part of 296 evaluable pts and toxicity data are tabulated below. The median follow-up is 2.2 years. None of the differences in CR rate, OS, or DFS are statistically significant. ADE (n=149) ADEP (n=147) CR 77% 78% CR - CBF AML 100% (21/21) 100% (25/25) CR With One Induction Course 89% 88% Induction Mortality 7% 7% Median OS (months) 21 20 Median DFS (months) 19 15 Median OS -pts ≤ 45 yrs (months) 29 Not Reached Median DFS pts ≤ 45 yrs (months) Not Reached 13 Grade 3 and 4 Toxicities Dysphagia/Esophagitis 6% 17% Stomatitis 9% 24% Bilirubin 6% 25% Rash 1% 10% Left Ventricular Function 2% 1% The high dose daunorubicin regimen was given safely, without excess induction mortality or cardiotoxicity. Hepatotoxicity due to ADEP consisted of generally reversible hyperbilirubinemia. PSC833 did not cause significant neurotoxicity. Compared with the induction regimen used in the previous group study in this pt population in which 474 pts received D 45 mg/m2 x 3 days and A 200 mg/m2 x 7 days without E or PSC833, (CALGB 9222, Blood2005; 105:3420), the CR rates in this study were higher (78% vs 72%), the number of pts achieving CR with 1 induction higher (89% vs. 77%), and the induction mortality comparable (7% vs 9%). These preliminary findings suggest that similar outcomes occur following an induction regimen using 2.25-fold higher D and 2.5-fold higher E doses (ADE) relative to a regimen employing a Pgp modulator (ADEP). PSC833 is known to delay the hepatic clearance of D and E. These data cannot distinguish between the possibilities that PSC833 augments the anti-leukemia activity of the lower dose ADE regimen or whether there is, in fact, no clinically meaningful dose-response relationship with D and E within the evaluated dose ranges. The findings of this trial to date suggest that Pgp-mediated drug efflux may not be the major or sole mechanism of drug resistance in AML or that PSC833 is an inadequate inhibitor of Pgp activity or both.

Description: We investigated the relative prognostic significance of cytogenetics in 635 adult acute myeloid leukemia (AML) patients 60 years of age or older treated on front-line protocols. Classification trees and tree-structured survival analysis (TSSA) were used to identify important cytogenetic groups, and their prognostic significance was then assessed in multivariable analysis (MVA). Overall, 48.5% achieved complete remission (CR); 6.6% survived at 5 years. Complex karyotypes with at least 3 abnormalities (complex ≥ 3) and a group including “rare aberrations” predicted lower CR rates (25% and 30%) versus other patients (56%). Compared with complex ≥ 3, the odds of CR were significantly higher for noncomplex karyotypes without rare aberrations on MVA. Cytogenetically, complex ≥ 5 predicted inferior disease-free survival on TSSA, remaining significant on MVA together with white blood cell count (WBC), sex, and age. For survival, complex ≥ 5, rare aberrations, and core-binding factor (CBF) abnormalities were prognostic (P 〈 .001), with 5-year survivals of 0%, 0%, and 19.4%, respectively, and 7.5% for remaining patients. Together with WBC, marrow blasts, sex, and age, the cytogenetic groups remained significant on MVA. In conclusion, pretreatment cytogenetics adds to other prognostic factors in older AML patients. Patients with complex ≥ 5 appear to benefit minimally from current treatment and are better suited for investigational therapy or supportive care. (Blood. 2006;108:63-73)