Therapy-related myeloid neoplasms (t-MN) are well-recognized complications of high-dose cytotoxic therapy (HDT), such as autologous stem cell transplantation (ASCT). Clonal marrow cytogenetic abnormalities (CMCA) in the setting of normal bone marrow pathology have also been reported after HDT, but their significance remains unclear. We retrospectively evaluated occurrences of CMCA and t-MN in 785 patients treated with HDT at Johns Hopkins University between 1997 and 2007. Most patients received ASCT, but 106 patients who received high-dose cyclophosphamide without ASCT were also included in this study, as this is our institutional standard for malignant and nonmalignant lymphoproliferative disorders in need of HDT. Twenty-two patients developed t-MN, with an estimated cumulative incidence of 3.5% at 4 years. Eleven patients developed isolated CMCA, either transient or persistent without pathologic evidence of t-MN. Altogether, only 20 of the patients with reported CMCA subsequently developed t-MN during the follow-up period. Therefore, in the absence of pathologic evidence of t-MN, CMCA should not be considered diagnostic of t-MN.
Treatment-related myeloid neoplasms; Clonal cytogenetic abnormalities; Chromosomal alterations in myeloid neoplasms
Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with poor outcomes with current therapies. Gemcitabine is the primary adjuvant drug used clinically, but its effectiveness is limited. In this study, our objective was to utilize a rationale-driven approach to identify novel biomarkers for outcome in patients with early-stage resected PDAC treated with adjuvant gemcitabine. Using a synthetic lethal screen in human PDAC cells, we identified 93 genes including 55 genes linked to DNA damage responses (DDR) that demonstrated gemcitabine sensitization when silenced, including CHD7 which functions in chromatin remodeling. CHD7 depletion sensitized PDAC cells to gemcitabine and delayed their growth in tumor xenografts. Moreover, CHD7 silencing impaired ATR-dependent phosphorylation of CHK1 and increased DNA damage induced by gemcitabine. CHD7 was dysregulated, ranking above the 90th percentile in differential expression in a panel of PDAC clinical specimens, highlighting its potential as a biomarker. Immunohistochemical analysis of specimens from 59 resected PDAC patients receiving adjuvant gemcitabine revealed that low CHD7 expression was associated with increased recurrence-free survival (RFS) and overall survival (OS), in univariate and multivariate analyses. Notably, CHD7 expression was not associated with RFS or OS for patients not receiving gemcitabine. Thus, low CHD7 expression was correlated selectively with gemcitabine sensitivity in this patient population. These results supported our rationale-driven strategy to exploit dysregulated DDR pathways in PDAC to identify genetic determinants of gemcitabine sensitivity, identifying CHD7 as a novel biomarker candidate to evaluate further for individualizing PDAC treatment.
Pancreatic Cancer; DNA Damage Response; CHD7; Biomarker; Gemcitabine; Replication Stress; ATR; CHK1
Mixed lineage kinase domain-like protein (MLKL) is a necrosome component mediating programmed necrosis that may be an important determinant of cancer cell death. The goal of the current study was to evaluate the prognostic value of MLKL expression in patients with pancreatic adenocarcinoma (PAC).
Tissue from 80 patients was collected from a prospectively maintained database of patients with PAC who underwent pancreaticoduodenectomy between January 2000 and October 2008. Immunohistochemistry analysis was performed and scored using an established scoring system. Kaplan-Meier survival curves were generated for recurrence-free survival (RFS) and overall survival (OS) for all patients and for patients receiving adjuvant chemotherapy. MLKL scores were correlated with RFS and OS using univariate and multivariate Cox regression analyses incorporating clinically relevant covariates.
The median age of the patients was 63 years and 53% were men. Low MLKL expression was associated with decreased OS (6 months vs 17 months; P=.006). In the subset of 59 patients who received adjuvant chemotherapy, low MLKL expression was associated with decreased RFS (5 months vs 15 months; P=.006) and decreased OS (6 months vs 19 months; P<.0001). On multivariate analysis, low MLKL expression was associated with poor OS in all patients (hazards ratio, 4.6 [95% confidence interval, 1.6-13.8]; P=.006) and in patients receiving adjuvant chemotherapy (hazards ratio, 8.1 [95% confidence interval, 2.2-29.2]; P=.002).
Low expression of MLKL is associated with decreased OS in patients with resected PAC and decreased RFS and OS in the subset of patients with resected PAC who receive adjuvant chemotherapy. The use of this biomarker in patients with PAC may provide important prognostic information.
mixed lineage kinase domain-like protein (MLKL); pancreatic cancer; biomarker; necrosis; necroptosis
Low vitamin D levels have been shown to be associated with primary hyperparathyroidism, but it is unclear whether vitamin D deficiency may be an etiologic factor in the development of primary hyperparathyroidism. To investigate this, we compared preoperative vitamin D levels of patients undergoing surgery for primary hyperparathyroidism with those of patients undergoing surgery for benign thyroid disease. With Institutional Review Board approval, data were collected prospectively on patients undergoing parathyroidectomy or thyroidectomy by one surgeon between March 2006 and July 2011. Patients were excluded if they underwent simultaneous thyroid and parathyroid surgery, had secondary or tertiary hyperparathyroidism, if no preoperative vitamin D level was measured, or if they took vitamin D supplements. Inclusion criteria were met by 219 patients who underwent parathyroidectomy and 186 patients who underwent thyroid surgery. Patient age, sex, race, and preoperative vitamin D levels (vitamin D 25-OH; normal, 32 to 100 pg/mL) were collected. Statistical analysis was performed using linear regression. Vitamin D levels were significantly lower in the parathyroid group compared with the thyroid group (23.8 vs 28.5 pg/mL; P <0.001). This difference was also observed after adjustment for age, sex, and race with a mean difference of 4.87 pg/mL (P <0.001). Statistically significant associations between lower vitamin D levels and patients younger than 50 years (P = 0.048), male sex (P = 0.03), and nonwhite race were identified (P < 0.001). Patients with primary hyperparathyroidism are more likely to have lower vitamin D levels than a control surgical population. Further study is needed to determine whether low vitamin D levels may be an etiologic factor associated with the development of hyperparathyroidism.
DNA methylation is an early event in bronchial carcinogenesis and increased DNA methyltransferase (DNMT)1 protein expression is a crucial step in the oncogenic transformation of epithelia. Here, we investigate the role of class I histone deacetylases (HDACs) 1–3 in the stabilization of DNMT1 protein and as a potential therapeutic target for lung cancer chemoprevention. Long-term exposure of immortalized bronchial epithelial cells (HBEC-3KT) to low doses of tobacco-related carcinogens led to oncogenic transformation, increased HDAC expression, cell cycle independent increased DNMT1 stability and DNA hypermethylation. Overexpression of HDACs was associated with increased DNMT1 stability and knockdown of HDACs reduced DNMT1 protein levels and induced DNMT1 acetylation. This suggests a causal relationship among increased class I HDACs levels, upregulation of DNMT1 protein, and subsequent promoter hypermethylation. Targeting of class I HDACs with valproic acid (VPA) was associated with reduced HDAC expression and a profound reduction of DNMT1 protein level. Treatment of transformed bronchial epithelial cells with VPA resulted in reduced colony formation, demethylation of the aberrantly methylated SFRP2 promoter and de-repression of SFRP2 transcription. These data suggest that inhibition of HDAC activity may reverse or prevent carcinogen induced transformation. Finally, immunohistochemistry on human lung cancer specimens revealed a significant increase in DNMT1, HDAC1, HDAC2, and HDAC3 expression, supporting our hypotheses that class I HDACs are mediators of DNMT1 stability.
In summary, our study provides evidence for an important role of class I HDACs in controlling the stability of DNMT1 and suggests that HDAC inhibition could be an attractive approach for lung cancer chemoprevention.
The primary purposes of Phase I cancer clinical trials are to determine the maximum tolerated dose (MTD) and the treatment schedule of a new drug. Phase I trials usually involve a small number of patients so that fully utilizing all toxicity information including time to event toxicity data is key to improving the trial efficiency and the accuracy of MTD estimation. Chen et al.  proposed a novel normalized equivalent toxicity score (NETS) system to fully utilize multiple toxicities per patient instead of a binary indicator of dose limiting toxicity (DLT). Cheung and Chappell  developed the time to toxicity event (TITE) approach to incorporate time to toxicity event data. Escalation with Overdose Control (EWOC) is an adaptive Bayesian Phase I design which can allow rapid dose escalation while controlling the probability of overdosing patients . In this manuscript, we use EWOC as a framework and integrate it with the NETS system and the TITE approach to develop an advanced Phase I design entitled EWOC-NETS-TITE. We have conducted simulation studies to compare its operating characteristics using selected derived versions of EWOC because EWOC itself has already been extensively compared with common Phase I designs . Simulation results demonstrate that EWOC-NETS-TITE can substantially improve the trial efficiency and accuracy of MTD determination as well as allow patients to be entered in a staggered fashion to significantly shorten trial duration. Moreover, user-friendly software for EWOC-NETS-TITE is under development.
Escalation with Overdose Control; Maximum Tolerated Dose; Multiple Toxicities; Quasi-continuous; Normalized Equivalent Toxicity Score; Toxicity Score System; Time to Toxicity Event; Phase I Clinical Trial
Poly (ADP) ribose polymerase (PARP) plays a key role in DNA repair and is highly expressed in small cell lung cancer (SCLC). We investigated the therapeutic impact of PARP inhibition in SCLC. In vitro cytotoxicity of veliparib, cisplatin, carboplatin, and etoposide singly and combined was determined by MTS in 9 SCLC cell lines (H69, H128, H146, H526, H187, H209, DMS53, DMS153, and DMS114). Subcutaneous xenografts in athymic nu/nu mice of H146 and H128 cells with relatively high and low platinum sensitivity, respectively, were employed for in vivo testing. Mechanisms of differential sensitivity of SCLC cell lines to PARP inhibition were investigated by comparing protein and gene expression profiles of the platinum sensitive and the less sensitive cell lines. Veliparib showed limited single-agent cytotoxicity but selectively potentiated (≥50% reduction in IC50) cisplatin, carboplatin, and etoposide in vitro in five of nine SCLC cell lines. Veliparib with cisplatin or etoposide or with both cisplatin and etoposide showed greater delay in tumor growth than chemotherapy alone in H146 but not H128 xenografts. The potentiating effect of veliparib was associated with in vitro cell line sensitivity to cisplatin (CC = 0.672; P = 0.048) and DNA-PKcs protein modulation. Gene expression profiling identified differential expression of a 5-gene panel (GLS, UBEC2, HACL1, MSI2, and LOC100129585) in cell lines with relatively greater sensitivity to platinum and veliparib combination. Veliparib potentiates standard cytotoxic agents against SCLC in a cell-specific manner. This potentiation correlates with platinum sensitivity, DNA-PKcs expression and a 5-gene expression profile.
Carboplatin; cisplatin; etoposide; PARP; SCLC; veliparib (ABT-888)
The current study was conducted to develop a multifactorial statistical model to predict the specific head and neck (H&N) tumor site origin in cases of squamous cell carcinoma confined to the cervical lymph nodes (“unknown primaries”).
The Surveillance, Epidemiology, and End Results (SEER) database was analyzed for patients with an H&N tumor site who were diagnosed between 2004 and 2011. The SEER patients were identified according to their H&N primary tumor site and clinically positive cervical lymph node levels at the time of presentation. The SEER patient data set was randomly divided into 2 data sets for the purposes of internal split-sample validation. The effects of cervical lymph node levels, age, race, and sex on H&N primary tumor site were examined using univariate and multivariate analyses. Multivariate logistic regression models and an associated set of nomograms were developed based on relevant factors to provide probabilities of tumor site origin.
Analysis of the SEER database identified 20,011 patients with H&N disease with both site-level and lymph node-level data. Sex, race, age, and lymph node levels were associated with primary H&N tumor site (nasopharynx, hypopharynx, oropharynx, and larynx) in the multivariate models. Internal validation techniques affirmed the accuracy of these models on separate data.
The incorporation of epidemiologic and lymph node data into a predictive model has the potential to provide valuable guidance to clinicians in the treatment of patients with squamous cell carcinoma confined to the cervical lymph nodes.
unknown primary; Surveillance; Epidemiology; End Results (SEER); cervical lymph nodes; predictive model; radiation
We present a pipeline to perform integrative analysis of mate-pair (MP) and paired-end (PE) genomic DNA sequencing data. Our pipeline detects structural variations (SVs) by taking aligned sequencing read pairs as input and classifying these reads into properly paired and discordantly paired categories based on their orientation and inferred insert sizes. Recurrent SV was identified from the discordant read pairs. Our pipeline takes into account genomic annotation and genome repetitive element information to increase detection specificity. Application of our pipeline to whole-genome MP and PE sequencing data from three multiple myeloma cell lines (KMS11, MM.1S, and RPMI8226) recovered known SVs, such as heterozygous TRAF3 deletion, as well as a novel experimentally validated SPI1 – ZNF287 inter-chromosomal rearrangement in the RPMI8226 cell line.
structural variations; multiple myeloma; whole-genome sequencing; variant detection
Aberrant promoter DNA methylation can serve as a predictive biomarker for improved clinical responses to certain chemotherapeutics. One of the major advantages of methylation biomarkers is the ease of detection and clinical application. In order to identify methylation biomarkers predictive of a response to a taxane-platinum based chemotherapy regimen in advanced NSCLC we performed an unbiased methylation analysis of 1,536 CpG dinucleotides in cancer-associated gene loci and correlated results with clinical outcomes.
We studied a cohort of 49 patients (median age 62 years) with advanced NSCLC treated at the Atlanta VAMC between 1999 and 2010. Methylation analysis was done on the Illumina GoldenGate Cancer panel 1 methylation microarray platform. Methylation data were correlated with clinical response and adjusted for false discovery rates.
Cav1 methylation emerged as a powerful predictor for achieving disease stabilization following platinum taxane based chemotherapy (p = 1.21E-05, FDR significance = 0.018176). In Cox regression analysis after multivariate adjustment for age, performance status, gender, histology and the use of bevacizumab, CAV1 methylation was significantly associated with improved overall survival (HR 0.18 (95%CI: 0.03–0.94)). Silencing of CAV1 expression in lung cancer cell lines(A549, EKVX)by shRNA led to alterations in taxane retention.
CAV1 methylation is a predictor of disease stabilization and improved overall survival following chemotherapy with a taxane-platinum combination regimen in advanced NSCLC. CAV1 methylation may predict improved outcomes for other chemotherapeutic agents which are subject to cellular clearance mediated by caveolae.
We performed a retrospective cohort analysis of 701 (533 White and 144 Black) patients with DLBCL treated at two referral centers in southern United States between 1981-2010. Median age of diagnosis for Blacks was 50 years vs. 57 years for Whites (p<0.001). A greater percentage of Blacks presented with elevated lactate dehydrogenase levels, B-symptoms, and performance status≥2. More Whites (8%) than Blacks (3%) had positive family history of lymphoma (p=0.048). There were no racial differences in the use of R-CHOP (52% Black vs. 47% White, p=0.73). While black race predicted worse survival among patients treated with CHOP (Hazard ratio [HR] 1.8, p<0.001), treatment with R-CHOP was associated with improved survival irrespective of race (HR 0.61, p=0.01). Future studies should examine biological differences that may underlie the observed racial differences in presentation and outcome.
Diffuse large B-cell lymphoma; Race; Disparities; Outcomes; CHOP regimen; R-CHOP regimen
Escalation with overdose control (EWOC) is a Bayesian adaptive design for selecting dose levels in cancer Phase I clinical trials while controlling the posterior probability of exceeding the maximum tolerated dose (MTD). EWOC has been used by clinicians to design many cancer Phase I clinical trials, see e.g [1-4]. However, this design treats the toxicity response as a binary indicator of dose limiting toxicity (DLT) and does not account for the number and specific grades of toxicities experienced by patients during the trial. Chen et al. (2010) proposed a novel toxicity score system to fully utilize all toxicity information using a normalized equivalent toxicity score (NETS). In this paper, we propose to incorporate NETS into EWOC using a quasi-Bernoulli likelihood approach to design cancer Phase I clinical trials. We call the design escalation with overdose control using normalized equivalent toxicity score (EWOC-NETS). Simulation results show that this design has good operating characteristics and improves the accuracy of MTD, trial efficiency, therapeutic effect, and overdose control relative to EWOC which is used as a representative of designs treating toxicity response as a binary indicator of DLT. We illustrate the performance of this design using real trial data in identifying the Phase II dose.
Escalation with Overdose Control; Maximum Tolerated Dose; Multiple Toxicities; Quasi-continuous; Normalized Equivalent Toxicity Score; Toxicity Score System
The preoperative diagnosis of thyroid nodules primarily depends upon fine needle aspiration (FNA) cytology. However, up to 25% of FNA samples have associated “suspicious or indeterminate”, but not diagnostic cytologic reports, resulting in difficulty deciding appropriate clinical management for these patients. We hypothesize that the use of molecular markers as an adjunct to FNA cytology can improve the distinction of benign from malignant nodules that have associated suspicious or indeterminate cytology.
Using microarray analysis, we previously identified and reported on 75 genes useful in the distinction of benign versus malignant thyroid nodules. In the present study, we have further validated the expression of 14 of these markers in a large number of thyroid samples by immunohistochemistry (IHC) analysis of 154 thyroid tumors and quantitative real-time RT-PCR (QRT-PCR) analysis of 95 FNA samples. Of the 154 tumors analyzed by IHC, 44 samples (29%) had associated suspicious or indeterminate FNA cytology.
Receiver operating characteristic using three-gene model, (HMGA2, MRC2, and SFN) analysis for the detection of malignant nodules resulted in areas under the curve (AUCs) of≥0.95 (80% sensitivity; 100% specificity) and≥0.84 (71% sensitivity; 84% specificity) for the IHC data in tumors, and QRT-PCR data in FNA samples, respectively.
Our results suggest that a three-gene model for the cytological diagnosis of indeterminate thyroid nodules is both feasible and promising. Implementation of this as an adjunct to thyroid cytology may significantly impact the clinical management of patients with suspicious or indeterminate thyroid FNA nodules.
The role of estrogen receptor alpha (ER) in breast cancer development and as a primary clinical marker for breast cancer prognosis is well documented. In this study, we identified the oncogenic protein TWIST1 (Twist), which is over-expressed in high-grade breast cancers, as a potential negative regulator of ER expression. Functional characterization of ER regulation by Twist was carried out using Twist low (MCF-7, T-47D) and Twist high (Hs 578T, MDA-MB-231, MCF-7/Twist) expressing cell lines. All Twist high cell lines exhibited low ER transcript and protein levels. By chromatin immunoprecipitation and promoter assays, we demonstrated that Twist could directly bind to E-boxes in the ER promoter and significantly down-regulate ER promoter activity in vitro. Functionally, Twist over-expression caused estrogen independent proliferation of breast cells and promoted hormone resistance to the selective estrogen receptor modulator (SERM) tamoxifen and selective estrogen receptor down-regulator (SERD) fulvestrant. Importantly, this effect was reversible on down-regulating Twist. Additionally, orthotopic tumors generated in mice using MCF-7/Twist cells were resistant to tamoxifen. These tumors had high vascular volume and permeability surface area as determined by magnetic resonance imaging (MRI). Mechanistically, Twist recruited DNA methyltransferase 3B (DNMT3B) to the ER promoter leading to a significantly higher degree of ER promoter methylation compared to parental cells. Furthermore, we demonstrated by co-immunoprecipitation that Twist interacted with histone deacetylase 1 (HDAC1) at the ER promoter, causing histone deacetylation and chromatin condensation, further reducing ER transcript levels. Functional re-expression of ER was achieved using demethylating agent 5-azacytidine and histone deacetylase inhibitor valproic acid. Finally, an inverse relationship was observed between Twist and ER expression in human breast tumors. In summary, the regulation of ER by Twist could be an underlying mechanism for loss of ER activity observed in breast tumors and may contribute to the generation of hormone resistant ER negative breast cancer.
Breast cancer; estrogen receptor; hormone resistance; TWIST1; Twist; transcriptional regulation; chromatin immunoprecipitation; co-immunoprecipitation; histone deacetylation; histone deacetylase 1 (HDAC1); methylation; DNA methyltransferase 3B (DNMT3B)
Isotonic Design using Normalized Equivalent Toxicity Score (ID-NETS) is a novel Phase I design that integrates the novel toxicity scoring system originally proposed by Chen et al.  and the original Isotonic Design proposed by Leung et al. . ID-NETS has substantially improved the accuracy of maximum tolerated dose (MTD) estimation and trial efficiency in the Phase I clinical trial setting by fully utilizing all toxicities experienced by each patient and treating toxicity response as a quasi-continuous variable instead of a binary indicator of dose limiting toxicity (DLT). To facilitate the incorporation of the ID-NETS method into the design and conduct of Phase I clinical trials, we have designed and developed a user-friendly software, ID-NETS©TM, which has two functions: 1) Calculating the recommended dose for the subsequent patient cohort using available completed data; and 2) Performing simulations to obtain the operating characteristics of a trial designed with ID-NETS. Currently, ID-NETS©TMv1.0 is available for free download at http://winshipbbisr.emory.edu/IDNETS.html.
Isotonic design; normalized equivalent toxicity score; maximum tolerated dose; dose limiting toxicity; cancer phase I clinical trial; software.
Cancer stem cells (CSCs) are functionally defined by their ability to self-renew and recapitulate tumors in the ectopic setting. They have been identified in a growing number of human malignancies and their association with poor clinical outcomes has suggested that they are a major factor in dictating clinical outcomes. Moreover, recent studies have demonstrated that CSCs may display other functional attributes, such as drug resistance and invasion and migration, that implicates a broad role in clinical oncology spanning initial tumor formation, relapse following treatment, and disease progression. Although our knowledge regarding the basic biology of CSCs continues to improve, a major issue remains proof that they are clinically relevant, and translation of the CSC hypothesis from the lab to the clinic is of paramount importance. We will review current evidence supporting the role of CSCs in clinical oncology and discuss potential barriers and strategies in designing trials examining CSC-targeting agents.
Cancer stem cells; Metastasis; Drug resistance; Antineoplastic agents
High-dose cyclophosphamide has long been used an anticancer agent, a conditioning regimen for hematopoietic stem cell transplantation and as potent immunosuppressive agent in autoimmune diseases including aplastic anemia. High-dose cyclophosphamide is highly toxic to lymphocytes but spares hematopoietic stem cells because of their abundant levels of aldehyde dehydrogenase, the major mechanism of cyclophosphamide inactivation. High dose cyclophosphamide therapy induces durable remissions in most patients with acquired aplastic anemia. Moreover, high-dose cyclophosphamide without hematopoietic stem cell rescue has shown activity in a variety of other severe autoimmune diseases. Here we review the history of cyclophosphamide as is applies to aplastic anemia (AA) and other autoimmune diseases. Included here are the historical data from early patients treated for AA as well as an observational retrospective study in a single tertiary care hospital. This latter component was designed to assess the safety and efficacy of high-dose cyclophosphamide therapy without stem cell rescue in patients with refractory autoimmune diseases. We analyzed fully the 140 patients with severe, progressive autoimmune diseases treated. All patients discussed here received cyclophosphamide, 50 mg/kg per day for 4 consecutive days. Response, relapse and overall survival were measured. Response was defined as a decrease in disease activity in conjunction with a decrease or elimination of immune modulating drugs. Relapse was defined as worsening disease activity and/or a requirement of an increase in dose of, or administration of new, immunosuppressive medications. Hematologic recovery occurred in all patients. The overall response rate of the was 95%, and 44% of those patients remain progression-free with a median follow up time of 36 (range 1–120) months for the 140 patients analyzed together. The overall actuarial and event free survival across all diseases at 60 months is 90.7% and 20.6%, respectively. High- dose cyclophosphamide without stem cell rescue is well-tolerated and induces a high rate of remissions in severe autoimmune diseases.
Stem Cell transplantation; Cyclophosphamide; Autoimmune diseases; Autoimmunity; Hematologic recovery
Though tyrosine kinase inhibitors have redefined the care of chronic myeloid leukemia (CML), these agents have not proved curative, likely due to resistance of the leukemia stem cells (LSC). While a number of potential therapeutic targets have emerged in CML, their expression in the LSC remains largely unknown. We therefore isolated subsets of CD34+ stem/progenitor cells from normal donors and from patients with chronic phase or blast crisis CML. These cell subsets were then characterized based on ability to engraft immunodeficient mice and expression of candidate therapeutic targets. The CD34+CD38− CML cell population with high aldehyde dehydrogenase (ALDH) activity was the most enriched for immunodeficient mouse engrafting capacity. The putative targets: PROTEINASE 3, SURVIVIN, and hTERT were expressed only at relatively low levels by the CD34+CD38−ALDHhigh CML cells, similar to the normal CD34+CD38−ALDHhigh cells and less than in the total CML CD34+ cells. In fact, the highest expression of these antigens was in normal, unfractionated CD34+ cells. In contrast, PRAME and WT1 were more highly expressed by all CML CD34+ subsets than their normal counterparts. Thus, ALDH activity appears to enrich for CML stem cells, which display an expression profile that is distinct from normal stem/progenitor cells and even the CML progenitors. Indeed, expression of a putative target by the total CD34+ population in CML does not guarantee expression by the LSC. These expression patterns suggest that PROTEINASE 3, SURVIVIN, and hTERT are not optimal therapeutic targets in CML stem cells; whereas PRAME and WT1 seem promising.
chronic myeloid leukemia; CML; leukemia stem cell; WT1; PRAME
Although the high mobility group A1 (HMGA1) gene is widely overexpressed in diverse cancers and portends a poor prognosis in some tumors, the molecular mechanisms that mediate its role in transformation have remained elusive. HMGA1 functions as a potent oncogene in cultured cells and induces aggressive lymphoid tumors in transgenic mice. Because HMGA1 chromatin remodeling proteins regulate transcription, HMGA1 is thought to drive malignant transformation by modulating expression of specific genes. Genome-wide studies to define HMGA1 transcriptional networks during tumorigenesis, however, are lacking. To define the HMGA1 transcriptome, we analyzed gene expression profiles in lymphoid cells from HMGA1a transgenic mice at different stages in tumorigenesis.
RNA from lymphoid samples at 2 months (before tumors develop) and 12 months (after tumors are well-established) was screened for differential expression of > 20,000 unique genes by microarray analysis (Affymetrix) using a parametric and nonparametric approach. Differential expression was confirmed by quantitative RT-PCR in a subset of genes. Differentially expressed genes were analyzed for cellular pathways and functions using Ingenuity Pathway Analysis. Early in tumorigenesis, HMGA1 induced inflammatory pathways with NFkappaB identified as a major node. In established tumors, HMGA1 induced pathways involved in cell cycle progression, cell-mediated immune response, and cancer. At both stages in tumorigenesis, HMGA1 induced pathways involved in cellular development, hematopoiesis, and hematologic development. Gene set enrichment analysis showed that stem cell and immature T cell genes are enriched in the established tumors. To determine if these results are relevant to human tumors, we knocked-down HMGA1 in human T-cell leukemia cells and identified a subset of genes dysregulated in both the transgenic and human lymphoid tumors.
We found that HMGA1 induces inflammatory pathways early in lymphoid tumorigenesis and pathways involved in stem cells, cell cycle progression, and cancer in established tumors. HMGA1 also dyregulates genes and pathways involved in stem cells, cellular development and hematopoiesis at both early and late stages of tumorigenesis. These results provide insight into HMGA1 function during tumor development and point to cellular pathways that could serve as therapeutic targets in lymphoid and other human cancers with aberrant HMGA1 expression.
Although previous studies have established a prominent role for HMGA1 (formerly HMG-I/Y) in aggressive human cancers, the role of HMGA2 (formerly HMGI-C) in malignant transformation has not been clearly defined. The HMGA gene family includes HMGA1, which encodes the HMGA1a and HMGA1b protein isoforms, and HMGA2, which encodes HMGA2. These chromatin binding proteins function in transcriptional regulation and recent studies also suggest a role in cellular senescence. HMGA1 proteins appear to participate in cell cycle regulation and malignant transformation, while HMGA2 has been implicated primarily in the pathogenesis of benign, mesenchymal tumors. Here, we show that overexpression of HMGA2 leads to a transformed phenotype in cultured lung cells derived from normal tissue. Conversely, inhibiting HMGA2 expression blocks the transformed phenotype in metastatic human non-small cell lung cancer cells. Moreover, we show that HMGA2 mRNA and protein are overexpressed in primary human lung cancers compared to normal tissue or indolent tumors. In addition, there is a statistically significant correlation between HMGA2 protein staining by immunohistochemical analysis and tumor grade (p < 0.001). Our results indicate that HMGA2 is an oncogene important in the pathogenesis of human lung cancer. Although additional studies with animal models are needed, these findings suggest that targeting HMGA2 could be beneficial therapeutically in lung and other cancers characterized by increased HMGA2 expression.
HMGA2; lung cancer; oncogene; antisense; transformation
Complete loss or deletion of the long arm of chromosome 5 is frequent in myelodysplastic syndrome (MDS) and acute myelogenous leukemia (AML). The putative gene(s) deleted and responsible for the pathogenesis of these poor prognosis hematological disorders remain controversial. This study is a comprehensive analysis of previously implicated and novel genes for epigenetic inactivation in AML and MDS. In 146 AML cases, methylation of CTNNA1 was frequent, and more common in AML patients with 5q deletion (31%) than those without 5q deletion (14%), while no methylation of other 5q genes was observed. In 31 MDS cases, CTNNA1 methylation was only found in high risk MDS (≥RAEB2), but not in low risk MDS (
del(5q); monosomy 5; CTNNA1; methylation; myelodysplastic syndrome; acute myelogenous leukemia; methylation; Progressive silencing; AML transformation
Although some reports have found increasing HLA disparity between donor and recipient to be associated with fewer relapses after allogeneic blood or marrow transplantation (BMT), this potential benefit has been offset by more graft-versus-host disease (GVHD) and nonrelapse mortality. However, the type of GVHD prophylaxis could influence the balance between GVHD toxicity and relapse. We analyzed the impact of greater HLA disparity on outcomes of a specific platform for nonmyeloablative, HLA-haploidentical transplantation. A retrospective analysis was performed on 185 patients with hematologic malignancies enrolled on three similar trials of nonmyeloablative, related donor, haploidentical BMT incorporating high-dose posttransplantation cyclophosphamide for GVHD prophylaxis. No significant association was found between the number of HLA mismatches (HLA-A, -B, -Cw, and -DRB1 combined) and risk of acute grade II–IV GVHD (hazard ratio .89, P = .68 for 3–4 versus fewer antigen mismatches). More mismatching also had no detrimental effect on event-free survival (on multivariate analysis, hazard ratio .60, P = .03 for 3–4 versus fewer antigen mismatches; hazard ratio .55, P = .03 for 3–4 versus fewer allele mismatches). Thus, greater HLA disparity does not appear to worsen overall outcomes after nonmyeloablative haploidentical BMT with high-dose posttransplantation cyclophosphamide.
Nonmyeloablative conditioning; allogeneic blood or marrow transplantation; graft-versus-host disease; human leukocyte antigens; cyclophosphamide
Although lung cancer is the leading cause of cancer death worldwide, the precise molecular mechanisms that give rise to lung cancer are incompletely understood. Here, we demonstrate that HMGA1 is an important oncogene that drives transformation in undifferentiated, large cell carcinoma. First, we show that the HMGA1 gene is overexpressed in lung cancer cell lines and primary human lung tumors. Forced overexpression of HMGA1 induces a transformed phenotype with anchorage-independent cell growth in cultured lung cells derived from normal tissue. Conversely, inhibiting HMGA1 expression blocks anchorage-independent cell growth in the H1299 metastatic, undifferentiated, large cell human lung carcinoma cells. We also demonstrate that the matrix metalloproteinase-2 (MMP-2) gene is a downstream target up-regulated by HMGA1 in large cell carcinoma cells. In chromatin immunoprecipitation experiments, HMGA1 binds directly to the MMP-2 promoter in vivo in large cell lung cancer cells, but not in squamous cell carcinoma cells. In large cell carcinoma cell lines, there is a significant, positive correlation between HMGA1 and MMP-2 mRNA. Moreover, interfering with MMP-2 expression blocks anchorage-independent cell growth in H1299 large cell carcinoma cells, indicating that the HMGA1-MMP-2 pathway is required for this transformation phenotype in these cells. Blocking MMP-2 expression also inhibits migration and invasion in the H1299 large cell carcinoma cells. Our findings suggest an important role for MMP-2 in transformation mediated by HMGA1 in large cell, undifferentiated lung carcinoma and support the development of strategies to target this pathway in selected tumors.
MMP-2; HMGA1; lung cancer; oncogene
Inflammation has been strongly implicated in prostate carcinogenesis, but the precise molecular mechanisms linking inflammation and carcinogenic DNA damage are not known. Induction of the polyamine catabolic enzyme, spermine oxidase (SMO) has been linked to increased reactive oxygen species (ROS) and DNA damage in human gastric and lung epithelial cells and suggest direct mechanistic links between inflammation, SMO activity, ROS production, and epithelial carcinogenesis that are likely relevant in prostate cancer.
Tissue microarrays consisting of matched normal and diseased specimens from patients diagnosed with prostate cancer, prostatic intraepithelial neoplasia (PIN), or proliferative inflammatory atrophy (PIA), as well as unaffected individuals, were stained for SMO expression and analyzed using image analysis techniques and TMAJ software tools.
Average SMO staining was significantly higher in prostate cancer and PIN tissues compared to patient-matched benign tissues. Benign tissues from prostate cancer, PIN, and PIA patients also exhibited significantly higher mean SMO expression versus tissues from prostate disease-free patients.
Tissues from patients diagnosed with prostate cancer and PIN exhibit, on average, locally increased SMO expression in regions of prostatic disease and higher overall SMO expression in prostatic epithelial cells compared to healthy individuals. Further studies are warranted to directly examine the role of SMO-produced ROS in prostate carcinogenesis.
prostate cancer; prostatic intraepithelial neoplasia; spermine oxidase; tissue microarrays; inflammation
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