Pediatric Hodgkin lymphoma: are we over-scanning our patients?

Despite the favorable outcome of most pediatric patients with Hodgkin lymphoma (HL), there is rising concern about risks of carcinogenesis from both diagnostic and therapeutic radiation exposure for patients treated on study protocols. Although previous studies have investigated radiation exposure during treatment, radiation from post-treatment surveillance imaging may also increase the likelihood of secondary malignancies. All diagnostic imaging examinations involving ionizing radiation exposure performed for surveillance following completion of therapy were recorded for 99 consecutive pediatric patients diagnosed with HL from 2000 to 2010. Cumulative radiation dosage from these examinations and the frequency of relapse detection by these examinations were recorded. In the first 2 years following completion of therapy, patients in remission received a median of 11 examinations (range 0-26). Only 13 of 99 patients relapsed, 11 within 5 months of treatment completion. No relapse was detected by 1- or 2-view chest radiographs (n = 38 and 296, respectively), abdomen/pelvis computed tomography (CT) scans (n = 211), or positron emission tomography (PET) scans alone (n = 11). However, 10/391 (2.6%) of chest CT scans, 4/364 (1.1%) of neck CT scans, and 3/47 (6.4%) of PET/CT scans detected relapsed disease. Thus, only 17 scans (1.3%) detected relapse in a total of 1358 scans. Mean radiation dosages were 31.97 mSv for Stage 1, 37.76 mSv for Stage 2, 48.08 mSv for Stage 3, and 51.35 mSv for Stage 4 HL. Approximately 1% of surveillance imaging examinations identified relapsed disease. Given the very low rate of relapse detection by surveillance imaging stipulated by current protocols for pediatric HL patients, the financial burden of the tests themselves, the high cure rate, and risks of second malignancy from ionizing radiation exposure, modification of the surveillance strategy is recommended.

Prdm14 initiates lymphoblastic leukemia after expanding a population of cells resembling common lymphoid progenitors.

Understanding the heterogeneous genetic mechanisms of tumor initiation in lymphoid leukemias (LL) will lead to improvements in prognostic classification and treatment regimens. In previous studies of mouse leukemias, we showed that retroviral insertion at the ecotropic viral insertion site 32 locus leads to increased expression of Prdm14, a pluripotency gene implicated in the self-renewal capacity of embryonic stem cells and the early stages of breast cancer. Here, we show that PRDM14 is also overexpressed in ∼25% of human lymphoid neoplasms, with increased frequencies in T-cell acute LL and hyperdiploid precursor B-cell acute LL. To test if Prdm14 overexpression could initiate leukemia, mice were transduced with bone marrow cells transfected with a Prdm14 expression vector. LLs developed in 96% of female mice and 42% of male mice. Before the onset of leukemia, differentiation of transduced cells was biased up to 1000-fold toward cells with features of common lymphoid progenitors (CLPs), and lymphoid differentiation showed a relative block at the pro-B stage. Microarray gene expression analysis of expanded CLP-like cells before the onset of leukemia demonstrated upregulation of genes involved in pluripotency, tumor initiation, early B-lineage commitment, Wnt/Ras signaling and the epithelial-to-mesenchymal transition. Among the dysregulated genes were imprinted genes and non-coding RNAs including Dlk1 and Meg3, which are also key pluripotency mediators. Heightened expression of the estrogen-dependent oncogene, Myb, in tumors suggests a basis for the increased frequency of cancer in female mice. These data provide the first direct evidence for the association of Prdm14 with cancer initiation in an in vivo mouse model and in human lymphoid malignancies, while suggesting mechanisms for Prdm14's mode of action.

Novel potential ALL low-risk markers revealed by gene expression profiling with new high-throughput SSH-CCS-PCR.

The current systems of risk grouping in pediatric acute lymphoblastic leukemia (ALL) fail to predict therapeutic success in 10-35% of patients. To identify better predictive markers of clinical behavior in ALL, we have developed an integrated approach for gene expression profiling that couples suppression subtractive hybridization, concatenated cDNA sequencing, and reverse transcriptase real-time quantitative PCR. Using this approach, a total of 600 differentially expressed genes were identified between t(4;11) ALL and pre-B ALL with no determinant chromosomal translocation. The expression of 67 genes was analyzed in different cytogenetic ALL subgroups and B lymphocytes isolated from healthy donors. Three genes, BACH1, TP53BPL, and H2B/S, were consistently expressed as a significant cluster associated with the low-risk ALL subgroups. A total of 42 genes were differentially expressed in ALL vs normal B lymphocytes, with no specific association with any particular ALL subgroups. The remaining 22 genes were part of a specific expression profile associated with the hyperdiploid, t(12;21), or t(4;11) subgroups. Using an unsupervised hierarchical cluster analysis, the discriminating power of these specific expression profiles allowed the clustering of patients according to their subgroups. These genes could help to understand the difference in treatment response and become therapeutical targets to improve ALL clinical outcomes.

CFFM4: a new member of the CD20/FcepsilonRIbeta family.

Proteins with transmembrane domains are classified in different families based on their structure, amino acid homology, and function. In this study, we report the identification, sequence, and expression profile of a new member of the CD20/FcepsilonRIbeta family, CD20/FcepsilonRIbeta family member 4 (CFFM4). The CFFM4 gene contains seven exons and six introns and is transcribed into an mRNA encoding a 240-amino acid protein with four hydrophobic regions. The CFFM4 protein shares a high degree of homology with the other members of the family, especially in the hydrophobic regions where several amino acids are conserved. However, the CFFM4 protein can be distinguished from the other members of the family based on the length of the second extracellular loop and the absence of an immunoreceptor tyrosine-based activation motif signal. Another distinct characteristic is that CFFM4 mRNA expression is not limited to the hematopoietic lineage. CFFM4 was detected by Northern dot blot in a variety of normal and cancerous tissues. CFFM4 expression was also compared in developmentally early hematopoietic human bone marrow CD34+ stem cells versus peripheral blood-derived CD14+ mature monocytes, in the undifferentiated versus differentiated myelomonocytic U937 cell line, and in acute myelogenous leukemia FAB1 versus FAB5. In each of these systems, cellular myelomonocytic differentiation correlated with an increase in CFFM4 mRNA expression. Such results indicate that CFFM4 is associated with mature cellular function in the monocytic lineage and like CD20 and FcepsilonRIbeta, it may be a component of a receptor complex involved in signal transduction.

Expression and localization of the CDC34 ubiquitin-conjugating enzyme in pediatric acute lymphoblastic leukemia.

Ubiquitin-dependent protein degradation impacts many cellular processes.However, the regulation of ubiquitin-conjugating enzymes (UBCs) in cancer is unknown. We find that the human CDC34 UBC protein is expressed at a 3-4 fold higher level (P < 0.001) in pediatric T cell than in pre-B-cell acute lymphoblastic leukemia (ALL) before treatment in two independent patient sets. The level of CDC34 mRNA was similar in both types of leukemia. CDC34 expression levels in normal resting T cells, B cells and activated T lymphocytes was comparable with pre-B-cell ALL. CDC34 protein (but not mRNA) was also increased in T-cell ALL compared with pre-B-cell ALL cell lines. The difference in expression was not attributable to mutation or associated with altered CDC34 stability. Immunohistochemistry and cellular fractionation reveals a heterogeneous CDC34 expression pattern including cells containing primarily cytoplasmic or nuclear protein. Thus, a feature of pediatric T-cell ALL is posttranscriptional up-regulation and heterogeneous localization of the human CDC34 UBC.

Differential expression of multiple unexpected genes during U937 cell and macrophage differentiation detected by suppressive subtractive hybridization.

OBJECTIVE: The objective of this study was to identify new markers of myelomonocytic differentiation using a sensitive technique that permits detection of rare differential gene expression. MATERIALS AND METHODS: [corrected] Suppressive subtractive hybridization (SSH) was performed between the human myelomonocytic U937 cell line and 1 alpha, 25-dihydroxyvitamin D3 and transforming growth factor beta 1 differentiated U937 cells. cDNA clones with significant increased expression in differentiated U937 cells over nondifferentiated U937 cells were characterized by sequencing. [corrected] The pattern of differential gene expression obtained by SSH was confirmed by cDNA Southern and Northern blots on the undifferentiated vs. differentiated U937 cells, and by reverse transcriptase polymerase chain reaction on undifferentiated human CD34(+) stem cells isolated from bone marrow vs. peripheral blood CD14(+) mature monocytes. RESULTS: Seven cDNAs never associated with in vitro U937 cell myelomonocytic differentiation (prolactin, 11-beta hydroxysteroid dehydrogenase [11 beta-HSD)] haptoglobin alpha (2FS)-beta precursor, GLIPR, RTVP, the RNA helicase P68, and spermidine-spermine N1-acetyltransferase) were identified. The first five of these genes previously were associated with immune function and the last two are important for intermediary metabolism. Differential expression was confirmed in CD34(+)/CD14(+) monocyte differentiation for all genes but 11 beta-HSD. CONCLUSIONS: We identified six new markers of U937 cell differentiation, which also are differentially expressed during normal human myelomonocytic differentiation.

Krüppel-associated boxes are potent transcriptional repression domains.

The Krüppel-associated box (KRAB) is a highly conserved, 75-aa region containing two predicted amphipathic alpha-helices. The KRAB domain is present in the amino-terminal regions of more than one-third of all Krüppel-class Cys2His2 zinc finger proteins and is conserved from yeast to man; however, its function is unknown. Here it is shown that the KRAB domain functions as a DNA binding-dependent transcriptional repressor when fused to a heterologous DNA-binding domain from the yeast GAL4 protein. A 45-aa segment containing one of the predicted KRAB amphipathic helices was necessary and sufficient for repression. Amino acid substitutions in the predicted helix abolished the repression function. These results assign a function, transcriptional repression, to the highly conserved KRAB box and define a minimal repression domain which may aid in identifying mechanisms of repression.

Structure and expression of the beta-platelet-derived growth factor receptor gene in human tumor cell lines.

Southern digests of DNA from 15 human connective tissue tumor-derived cell lines (and cell strains) and from peripheral blood lymphocytes from normal volunteers were prepared to compare structure of the beta-platelet-derived growth factor receptor gene in normal and tumor-derived cells. The tumor-derived samples had no alterations in gene structure, nor was gene amplification evident. Two restriction fragment length polymorphisms were detected within the beta-receptor gene. Expression of the alpha- and beta-platelet-derived growth factor receptor was quantified using Northern blots. Expression was not tumor-type specific. For example, one rhabdomyosarcoma cell line expressed only the alpha-receptor, whereas two others expressed the beta. In contrast, a human fibroblast cell strain expressed both receptor types. Alterations in receptor expression may be a result of the tumorigenic process.