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Patients with hematologic malignancies not in remission before allogeneic hematopoietic stem cell transplantation (HSCT) have a poor prognosis. To improve the antitumor activity of conditioning, we combined clofarabine with myeloablative doses of busulfan in a phase 1/2 study in nonremission hematologic malignancies. Forty-six patients were enrolled, including 31 patients with nonremission acute myelogenous leukemia (AML). Patients had a median age of 53 years, with a median comorbidity index of 3. Donors were unrelated, HLA mismatched, or both in 59% of patients. Common grade III to IV nonhematologic toxicities included transient transaminitis (50%), mucositis (24%), hand-foot syndrome (13%), transient hypoxia (13%), nausea/vomiting (9%), and diarrhea (9%). All patients engrafted. Complete remission was achieved in 80% of all patients by day +30 and in 100% of AML patients without prior hematopoietic stem cell transplantation. Two-year nonrelapse mortality for all patients was 31%, and overall survival was 28%. In AML, the overall survival was 48% at 1 year and 35% at 2 years. These data suggest that clofarabine combined with myeloablative doses of busulfan is well tolerated, secures engraftment, and possesses significant antitumor activity, particularly in nonremission AML. This study is registered at www.ClinicalTrials.gov under identifier NCT00556452.

Background: Primary thyroid lymphoma is an uncommon neoplasm frequently associated with lymphocytic thyroiditis (LT). Once the pathologic diagnosis of primary thyroid lymphoma is established, imaging plays an important role in tumor staging and evaluating treatment response. The present case discusses the role of fluorodeoxyglucose positron emission tomography (18F-FDG PET)/computed tomography (CT) in this clinical setting along with the potential diagnostic challenges., Patient Findings: A 44-year-old man with a history of LT and hypothyroidism presented with an enlarging goiter. Initial imaging evaluation showed markedly enlarged gland with bilateral cervical and mediastinal adenopathy. Histopathologic evaluation confirmed the diagnosis of primary thyroid lymphoma on a background of LT. An 18F-FDG PET/CT revealed increased uptake in the gland and lymph nodes. Follow-up 18F-FDG PET/CT after chemotherapy showed interval decrease in FDG uptake in the thyroid gland associated with interval decrease in the size and metabolic activity of the cervical and superior mediastinal lymph nodes., Conclusions: The frequent association of LT with primary thyroid lymphoma and the overlap of their clinical and pathologic findings pose a significant diagnostic challenge. While other imaging techniques are helpful in evaluating anatomic local and regional extent of primary thyroid lymphoma, 18F-FDG PET/CT can be of an added value in evaluating its metabolic activity and detecting regional and distant disease as well as in assessing response to treatment.

Genomic aberrations are of predominant importance to the biology and clinical outcome of patients with chronic lymphocytic leukemia (CLL), and FISH-based genomic risk classifications are routinely used in clinical decision making in CLL. One of the known limitations of CLL FISH is the inability to comprehensively interrogate the CLL genome for genomic changes. In an effort at overcoming the existing limitations in CLL genome analysis, we have analyzed high-purity DNA isolated from FACS-sorted CD19(+) cells and paired CD3(+) or buccal cells from 255 patients with CLL for acquired genomic copy number aberrations (aCNAs) with the use of ultra-high-density Affymetrix SNP 6.0 arrays. Overall, ≥ 2 subchromosomal aCNAs were found in 39% (100 of 255) of all cases analyzed, whereas ≥ 3 subchromosomal aCNAs were detected in 20% (50 of 255) of cases. Subsequently, we have correlated genomic lesion loads (genomic complexity) with the clinical outcome measures time to first therapy and overall survival. With the use of multivariate analyses incorporating the most important prognostic factors in CLL together with SNP 6.0 array-based genomic lesion loads at various thresholds, we identify elevated CLL genomic complexity as an independent and powerful marker for the identification of patients with aggressive CLL and short survival.

Purpose: The chromosomal deletion 11q affects biology and clinical outcome in chronic lymphocytic leukemia (CLL) but del11q-deregulated genes remain incompletely characterized., Experimental Design: We have employed integrated genomic profiling approaches on CLL cases with and without del11q to identify 11q-relevant genes., Results: We have identified differential expression of the insulin receptor (INSR) in CLL, including high-level INSR expression in the majority of CLL with del11q. High INSR mRNA expression in 11q CLL (∼10-fold higher mean levels than other genomic categories) was confirmed by quantitative PCR in 247 CLL cases. INSR protein measurements in 257 CLL cases through flow cytometry, compared with measurements in normal CD19(+) B cells and monocytes, confirmed that a subset of CLL aberrantly expresses high INSR levels. INSR stimulation by insulin in CLL cells ex vivo resulted in the activation of canonical INSR signaling pathways, including the AKT-mTOR and Ras/Raf/Erk pathways, and INSR activation partially abrogated spontaneous CLL cell apoptosis ex vivo. Higher INSR levels correlated with shorter time to first therapy and shorter overall survival (OS). In bivariate analysis, INSR expression predicted for rapid initial disease progression and shorter OS in ZAP-70-low/negative CLL. Finally, in multivariate analysis (ZAP-70 status, IgV(H) status, and INSR expression), we detected elevated HRs and trends for short OS for CLL cases with high INSR expression (analyzed inclusive or exclusive of cases with del11q)., Conclusions: Our aggregate biochemical and clinical outcome data suggest biologically meaningful elevated INSR expression in a substantial subset of all CLL cases, including many cases with del11q., (©2011 AACR.)

Genomic aberrations are of predominant importance to the biology and clinical outcome of patients with acute myelogenous leukemia (AML), and conventional karyotype-based risk classifications are routinely used in clinical decision making in AML. One of the known limitations of cytogenetic analysis is the inability to detect genomic abnormalities less than 5 Mb in size, and it is currently unclear whether overcoming this limitation with high-resolution genomic single-nucleotide polymorphism (SNP) array analysis would be clinically relevant. Furthermore, given the heterogeneity of molecular mechanisms/aberrations that underlie the conventional karyotype-based risk classifications, it is likely that further refinements in genomic risk prognostication can be achieved. In this study, we analyzed flow cytometer-sorted, AML blast-derived, and paired, buccal DNA from 114 previously untreated prospectively enrolled AML patients for acquired genomic copy number changes and loss of heterozygosity using Affymetrix SNP 6.0 arrays, and we correlated genomic lesion load and specific chromosomal abnormalities with patient survival. Using multivariate analyses, we found that having ≥ 2 genomic lesions detected through SNP 6.0 array profiling approximately doubles the risk of death when controlling for age- and karyotype-based risk. Finally, we identified an independent negative prognostic impact of p53 mutations, or p53 mutations and 17p-loss of heterozygosity combined on survival in AML.

Purpose: This study was conducted to identify novel genes with importance to the biology of adult acute myelogenous leukemia (AML)., Experimental Design: We analyzed DNA from highly purified AML blasts and paired buccal cells from 95 patients for recurrent genomic microdeletions using ultra-high density Affymetrix single nucleotide polymorphism 6.0 array-based genomic profiling., Results: Through fine mapping of microdeletions on 17q, we derived a minimal deleted region of approximately 0.9-Mb length that harbors 11 known genes; this region includes Neurofibromin 1 (NF1). Sequence analysis of all NF1 coding exons in the 11 AML cases with NF1 copy number changes identified acquired truncating frameshift mutations in two patients. These NF1 mutations were already present in the hematopoetic stem cell compartment. Subsequent expression analysis of NF1 mRNA in the entire AML cohort using fluorescence-activated cell sorting sorted blasts as a source of RNA identified six patients (one with a NF1 mutation) with absent NF1 expression. The NF1 null states were associated with increased Ras-bound GTP, and short hairpin RNA-mediated NF1 suppression in primary AML blasts with wild-type NF1 facilitated colony formation in methylcellulose. Primary AML blasts without functional NF1, unlike blasts with functional NF1, displayed sensitivity to rapamycin-induced apoptosis, thus identifying a dependence on mammalian target of rapamycin (mTOR) signaling for survival. Finally, colony formation in methylcellulose ex vivo of NF1 null CD34+/CD38- cells sorted from AML bone marrow samples was inhibited by low-dose rapamycin., Conclusions: NF1 null states are present in 7 of 95 (7%) of adult AML and delineate a disease subset that could be preferentially targeted by Ras or mammalian target of rapamycin-directed therapeutics.

Chronic lymphocytic leukemia (CLL) has a variable clinical course. Presence of specific genomic aberrations has been shown to impact survival outcomes and can help categorize CLL into clinically distinct subtypes. We studied 178 CLL patients enrolled in a prospective study at the University of Michigan, of whom 139 and 39 were previously untreated and previously treated, respectively. We obtained unbiased, high-density, genome-wide measurements of subchromosomal copy number changes in highly purified DNA from sorted CD19(+) cells and buccal cells using the Affymetrix 50kXbaI SNP array platform (Santa Clara, CA). Genomic complexity scores were derived and correlated with the surrogate clinical end points time to first therapy (TTFT) and time to subsequent therapy (TTST): measures of disease aggressiveness and/or therapy efficaciousness. In univariate analysis, progressively increasing complexity scores in previously untreated CLL patients identified patients with short TTFT at high significance levels. Similarly, TTST was significantly shorter in pretreated patients with high as opposed to low genomic complexity. In multivariate analysis, genomic complexity emerged as an independent risk factor for short TTFT and TTST. Finally, algorithmic subchromosomal complexity determination was developed, facilitating automation and future routine clinical application of CLL whole-genome analysis.

We describe longitudinal diffusion-weighted MRI findings of sub-acute leukoencephalopathy following methotrexate therapy in a 24-year-old man diagnosed with pre-B-cell acute lymphoblastic leukemia (ALL), presenting with right-sided paralysis and aphasia after second consolidation with intrathecal triple-drug therapy given intrathecally. This case demonstrates the value of DWI in evaluation and diagnosis of sub-acute toxic leukoencephalopathy in patients being treated with methotrexate. The longitudinal follow up DWI findings suggest reversible metabolic derangement rather than ischemia as the cause of these findings.

Inhibitors of the MDM2-p53 interaction are actively being developed as anti-cancer agents. Drug-induced interference with the MDM2 E3 ligase function or with MDM2 protein-protein interactions abrogates tonic suppression and destruction of the p53 protein; consequently, p53 steady state levels rise resulting in the induction of p53-dependent anti-proliferative and pro-apoptotic genes. Some cancerous cells harboring wild type p53 respond to MDM2 inhibitor-induced elevated p53 protein levels with apoptotic cell death while non-malignant cells, for poorly understood reasons, appear relatively resistant. Deciphering the mechanisms of resistance or susceptibility to MDM2 inhibitor-induced cancer cell death is of significant importance for the clinical development and applications of MDM2 inhibitory compounds and serves to illuminate aspects of MDM2 and p53 biology. Using data from ex vivo MDM2 inhibitor treatment of a large cohort of molecularly highly characterized CLL cases, we were able to demonstrate the central role of p53 status as a determinant of resistance in this common leukemia. In the context of these experimental findings, we summarize pertinent knowledge of the biology of p53, MDM2, p53 target genes and MDM2 binding proteins. Finally, using data from a large SNP-array-based high-density genomic profiling study in CLL, we summarize the genomic copy number and allele status for important p53 effector genes as well as for MDM2 binding/target proteins, thus demonstrating the power of high resolution genomic analysis in support of targeted drug development.

Chronic lymphocytic leukemia (CLL) is a biologically heterogeneous illness with a variable clinical course. Loss of chromosomal material on chromosome 13 at cytoband 13q14 is the most frequent genetic abnormality in CLL, but the molecular aberrations underlying del13q14 in CLL remain incompletely characterized. We analyzed 171 CLL cases for loss of heterozygosity and subchromosomal copy loss on chromosome 13 in DNA from fluorescence-activated cell sorting-sorted CD19(+) cells and paired buccal cells using the Affymetrix XbaI 50k SNP array platform. The resulting high-resolution genomic maps, together with array-based measurements of expression levels of RNA in CLL cases with and without del13q14 and quantitative PCR-based expression analysis of selected genes, support the following conclusions: (a) del13q14 is heterogeneous and composed of multiple subtypes, with deletion of Rb or the miR15a/miR16 loci serving as anatomic landmarks, respectively; (b) del13q14 type Ia deletions are relatively uniform in length and extend from breakpoints close to the miR15a/miR16 cluster to a newly identified telomeric breakpoint cluster at the approximately 50.2 to 50.5 Mb physical position; (c) LATS2 RNA levels are approximately 2.6-fold to 2.8-fold lower in cases with del13q14 type I that do not delete Rb, as opposed to del13q14 type II or all other CLL cases; (d) PHLPP RNA is absent in approximately 50% of CLL cases with del13q14; and (e) approximately 15% of CLL cases display marked reductions in miR15a/miR16 expression that are often but not invariably associated with bi-allelic miR15a/miR16 loss. These data should aid future investigations into biological differences imparted on CLL by different del13q14 subtypes.

Chronic lymphocytic leukemia (CLL) is the most common leukemia in the Western world and remains incurable with conventional therapies. Patients with relapsed or resistant CLL have a significantly shortened lifespan. MDM2 inhibitors have been developed and may have significant potential in the treatment of CLL. Clinical development of these compounds would be aided through knowledge of molecular predictors of activity. To understand determinants of sensitivity or resistance to MDM2 inhibitor therapy in CLL, we comprehensively analyzed a large cohort of CLL patient-derived samples for response to MDM2 inhibition and correlated these responses with clinically important biomarkers. Furthermore, we employed high-density single nucleotide polymorphism (SNP) arrays to analyze genomewide changes of copy number and allele status, including that of p53. The results of these studies conclusively demonstrate that p53 status is the major determinant of response to MDM2 inhibitors in CLL. Additional defects in the p53 regulatory cascade do not appear operational in this leukemia. Further, we identify a novel subgroup of patients with CLL with early progressive disease that appears particularly sensitive to MDM2 inhibitor treatment. These data provide definitive evidence for target-specific and predictive activity and a rationale to proceed with this potentially important class of compounds in the treatment of CLL.

Imatinib was the first treatment for chronic myeloid leukemia (CML) that specifically targeted the causative BCR-ABL oncoprotein, and represented a major therapeutic advance in this disease; however, some patients develop resistance or intolerance. Resistance can be classified as BCR-ABL-dependent (e.g., mutation in the BCR-ABL gene) or BCR-ABL-independent (alternative pathways of disease progression, e.g., SRC-family tyrosine kinases). The investigation of therapeutic options post-imatinib failure resulted in the development and regulatory approval of dasatinib, a BCR-ABL and SRC-family kinase inhibitor. Dasatinib is active across all phases of CML and Philadelphia chromosome-positive acute lymphoblastic leukemia, and demonstrates activity in almost all imatinib-resistant mutations. Other therapeutic options are also under investigation, with nilotinib being the most clinically advanced. Nilotinib is an analog of imatinib with similar multiple kinase targets, but without inhibition of SRC, and reduced in vitro activity against BCR-ABL P-loop mutations compared with dasatinib. Similar to dasatinib, nilotinib has no activity against T315I mutations. The availability of dasatinib and development of other tyrosine kinase inhibitors provide positive prospects for patients with imatinib-resistant or -intolerant CML. Here, we discuss several of these new strategies for treating patients after imatinib failure.

Polycyclic aromatic hydrocarbons (PAH) are a well-characterized group of mutagens and carcinogens. Benzo(a)pyrene [B(a)P], the best known compound in the group, exerts its genotoxic activity following metabolic activation, when it acquires the properties of an electrophilic reagent that is capable of interacting with DNA. Reactive oxygen species (ROS) can remerge during the PAH metabolic activation. Because of their antioxidant activity, vitamins C and E are thought to act as antimutagenic agents. We designed an in vitro protocol to study the potential protective effect of vitamins C and E toward B(a)P-induced DNA damage. In this study, we examined peripheral blood lymphocytes obtained from healthy nonsmoking female volunteers, 22 to 25 years of age. The cells were exposed in vitro to 1 microM B(a)P in the presence of 40 microM or 100 microM of vitamin C or, alternatively, to 30 microM or 100 microM of vitamin E. The B(a)P-induced DNA damage and repair were estimated as the generation and removal of single-strand DNA breaks measured by the alkaline version of the single-cell gel electrophoresis (comet) assay. The protective effect of vitamins C and E was demonstrated when the vitamins were applied simultaneously with or after the B(a)P. The background level of DNA damage in the presence of vitamins C and E was lower than in the system without the vitamins. The experiments were conducted according to various protocol schemes of the vitamin treatment and the results offer additional evidence of the antigenotoxic activity of vitamins C and E. The vitamin activity does not appear to be connected with the steps in metabolic activation or DNA repair. It seems that both vitamins act as competitors of DNA molecule in reaction with the reactive oxygen species.