Emerging therapies for acute myeloid leukemia: a qualitative case study

Treatment of Acute Myeloid Leukemia

As a person ages, his/her prospect of contracting Acute Myeloid Leukemia (AML)

increases. Children and adults of any ages, however, may develop AML This qualitative study asserts that the potential for development of significant improvement of therapies for AML depends on continuing studies such as the three which this qualitative case study investigates.

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Context of the Problem

Organization of the Study

TREATMENT of ACUTE MYELOID LEUKEMIA

"The overall cure rate for childhood [acute lymphocytic leukemia] ALL

is now approximately 75-80%; however, for [acute myelogenous leukemia] AML

the cure rate is between 40 and 45%"

(Belson, Kingsley, and Holmes, para. 6)

Context of the Problem

Acute Myeloid Leukemia (AML)

Leukemia currently maintains the status of being one of the 10 leading causes of cancer deaths in the United States. Although chemotherapy or allogeneic bone marrow transplantation, may contribute to a durable survival for a number of leukemia patients, adults with relapsed and/or refractory leukemias do not hold a satisfactory survival record. The number of Americans projected to be diagnosed with acute myelogenous leukemia (AML), a cancer of the blood and bone marrow, in the United States (U.S.) during 2008 was approximately 13,290. The prospect of contracting AML increases as a person ages, albeit children and adults of any age may develop AML (Detailed Guide: Leukemia). This thesis asserts that the potential for development of significant improvement of therapies for AML depends on continuing studies such as the three which this qualitative case study investigates.

In adults and children, AML typically worsens rapidly when not treated. In adults, whether the reason is due to their older age, the absence of a donor, or the refractory/progressive disease, the majority of leukemia patients may not have the option for allogeneic bone marrow, Ken Shiozawa, Takeo Nakanishi, Ming Tan, Hong-Bin Fang, Wen-chyi Wang, Martin J. Edelman, David Carlton, Ivana Gojo, Edward a. Sausville and Douglas D. Ross report in "Preclinical Studies of Vorinostat (Suberoylanilide Hydroxamic Acid) Combined with Cytosine Arabinoside and Etoposide for Treatment of Acute Leukemias," Study I, one of the three studies this thesis examines.

In Study II, "Nuclear Factor- B Modulation in Patients Undergoing Induction Chemotherapy for Acute Myelogenous Leukemia," the second study this thesis explores, Roger K. Strair, Mecide Gharibo, Dale Schaar, Arnold Rubin, Jonathan Harrison, Joseph Aisner, Hsin-Ching Lin5, Yong Lin, Lauri Goodell, Monika Anand, Binaifer Balsara, Liesel Dudek, Arnold Rabson and Daniel J. Medina investigated whether standard anti-inflammatory agents modulate AML cell nuclear NF-B when administered in conjunction with induction chemotherapy. Results from the study by Strair, et al. indicate the need warrants more follow-up study for determining the effects of NF-B modulation on clinical end points.

The researcher notes that in Study III, the third study this study explores, "Preclinical Studies of Vorinostat (Suberoylanilide Hydroxamic Acid) Combined with Cytosine Arabinoside and Etoposide for Treatment of Acute Leukemias," that the authors, Ken Shiozawa, Takeo Nakanishi, Ming Tan, Hong-Bin Fang, Wen-chyi Wang, Martin J. Edelman, David Carlton, Ivana Gojo, Edward a. Sausville and Douglas D. Ross, utilized an experimental design. Shiozawa, et al. appraised "combining cytosine arabinoside [1-?-D-arabinofuranosylcytosine (ara-C)] and/or etoposide with vorinostat for use in the treatment of acute leukemias" (Abstract). The assessment of drug combination effects by Shiozawa, et al. ultimately proffered a preclinical rationale for phase I trials of the sequential combination of vorinostat followed by ara-C and etoposide in patients evidencing advanced or refractory leukemias.

Statement of the Problem

While approximately one in five children with leukemia has AML, according to the Leukemia & Lymphoma Society (LLS), adult AML constitutes the most common type of acute leukemia in adults. The Leukemia & Lymphoma Society stresses it to be vital that acute leukemia be treated right away. At this time, no "cure" exists. The prognosis for the AML patient, as well as his/her treatment options depend on, but may not be limited to:

The age of the patient.

The subtype of AML.

Whether the patient received chemotherapy in the past to treat a different cancer.

Whether there is a history of a blood disorder such as myelodysplastic syndrome.

Whether the cancer has spread to the central nervous system.

Whether the cancer has been treated before or recurred (come back).

(Lukemia & Lymphoma…)

Significance of the Study

The fact that the overall cure rate for AML totals only 40 and 45%, approximately half, the rate for ALL (Belson, Kingsley, and Holmes), the researcher asserts, contributes to this study's claim to merit a "significant" status. In addition, as leukemia currently maintains the status of being one of the 10 leading causes of cancer deaths in the United States, the researcher contends that this thesis which explores the potential for development of significant improvement of therapies for AML, proves significant as it contributes to the understanding of possible advancements toward improved treatments.

The American Cancer Society notes the following key statistics regarding AML:

About 44,270 new cases of leukemia will be diagnosed in the United States during 2008.

About 8,820 deaths from AML will occur in the United States during 2008, and almost all will be in adults.

Acute myeloid leukemia is generally a disease of older people and is rare before the age of 40. The average age of a patient with AML is about 67 years.

AML is slightly more common among men than among women. The lifetime risk of getting AML for the average man is about 1 in 225; for the average woman the risk is about 1 in 300. (Detailed Guide: Leukemia).

Study Rationale

Current clinical concerns regarding AML, particularly regarding the low overall cure rate, along with the disease's high rate of new cases and deaths, noted in the previous section, factor into the researcher's rationale for choosing to focus on this disease. During the course of this thesis, the researcher relates a number of findings from examining three contemporary clinical studies related to AML. In turn, the researcher, as well as readers will increase understanding of this disease. Ultimately, the researcher contends, this research effort will serve as a source for consideration as a credible resource by other researchers and encourage even more research relating to treatment of AML.

Research Questions

For this study's research questions, the researcher drew/adapted the following three questions from the three studies explored during this thesis. These three questions double as the research questions for this study effort.

1. Does C/EBP mRNA serve as a target for miRNA-124? (Hackanson, et al.).

2. Do standard anti-inflammatory agents modulate AML cell nuclear NF-B when administered in conjunction with induction chemotherapy? (Strair, et al.).

3. How affective is the practice of "combining cytosine arabinoside [1-?-D-arabinofuranosylcytosine (ara-C)] and/or etoposide with vorinostat for use in the treatment of acute leukemias"? (Shiozawa Abstract).

Research Design and Methodology

Case Studies (2008) asserts that a case study research design, a form of qualitative descriptive research, provides the researcher with a blueprint for him/her to utilize to build his/her study. As the study examines "an individual or small participant pool, it draws conclusions only about that participant or group and only in that specific context" (Case Studies 2008, Introduction and Definition section). To complement this thesis which investigates treatment for AML, the researcher examined three clinical studies related to AML.

Organization of the Study

Chapter I: Introduction

This study's first chapter, Chapter I presents the background of the phenomenon, AML and introduces the focus for the thesis. Chapter I also denotes s the three clinical studies being expounded during this study effort.

Chapter II: Review of the Literature

Chapter II, the literature review relates the three studies the researcher chose to foucs on during this thesis. This chapter also presents additional information the researcher accessed for this study. The researcher subscribed to the American Association for Cancer Research Web site (http://cancerres.aacrjournals.org/) to access the three primary studies utilized as the foundation for this study's focus.

Chapter III: Methodology

Chapter III of this thesis mirrors the methodology the researcher used to implement this particular qualitative case study focusing on AML.

Chapter IV: Analysis

Chapter IV of this thesis relates numerous findings the researcher dissects from the three clinical studies. Chapter V: Discussion, Conclusion and Recommendations

Chapter V reviews the study scenario, recounts a number of the findings from the clinical studies, and reiterates relevant points addressing the three research questions regarding AML treatments. At the end of this study/chapter, the researcher proffers several recommendations, relating to the treatment of AML, for future researchers to consider for further research projects. The researcher also notes any experiences that could serve as lessons for future study opportunities.

Aims and Objectives

The primary aim for the study is to examine three clinical studies, noted earlier in this study, relating to the treatment of AML.

Objective 1

Access three specific clinical studies relating the treatment of AML.

Objective 2

Access additional literature which relates credible information relating to the treatment of AML.

Objective 3

Utilize the data accessed from the three studies to compile with informatio retrieved from the other sources to address the three research questions the researcher developed for this study's focus.

Conclusion

Despite the depressing figures embodied in the quote introducing this thesis, that: "The overall cure rate for AML…is between 40 and 45%" (Belson, Kingsley, and Holmes, para. 6), data/information related during the next chapter, the Literature Review, will contain a semblance of hope. Hope for the potential development of significant improvement of therapies for AML, the researcher projects, albeit, depends on continuing studies such as the three noted in/by this qualitative case study, along with this present one the researcher implements to explore those three.

CHAPTER II

LITERATURE REVIEW

"Acute myeloid leukemia (AML) goes by many names, including acute myelocytic leukemia, acute myelogenous leukemia, acute granulocytic leukemia, and acute non-lymphocytic leukemia"

(Detailed Guide: Leukemia).

The word "acute" in acute myeloid leukemia refers to the fact the leukemia may progress at a rapid rate, and if/when the disease is not treated, within a few months, it would likely prove to be fatal. AML begins to develop in cells that traditionally develop into various types of blood cells. Most AML cases develop from cells that would transform into white blood cells (other than lymphocytes), however, a number AML cases develop in other kinds of blood-forming cells. AML starts to develop in the bone marrow, albeit, in the majority of cases it rapidly moves into the blood. At times, it may spread to other body parts, "including the lymph nodes, liver, spleen, central nervous system (brain and spinal cord), and testes" (Detailed Guide: Leukemia). Some other cancer types that may start in these organs and/or elsewhere, and then spread to the bone marrow are not leukemia.

Information presented in this literature review chapter, the researcher notes, "names" or reports a number of relevant points regarding AML. The three clinical studies presented in this study's introduction relate particularly relevant data regarding treatments of/for AML. The following three studies the researcher examines during this thesis include:

1. Study I: "Preclinical Studies of Vorinostat (Suberoylanilide Hydroxamic Acid) Combined with Cytosine Arabinoside and Etoposide for Treatment of Acute Leukemias" (Shiozawa, et al.).

2. Study II: "Nuclear Factor- B Modulation in Patients Undergoing Induction Chemotherapy for Acute Myelogenous Leukemia" (Strair, et al.).

3. Study III: "Preclinical Studies of Vorinostat (Suberoylanilide Hydroxamic Acid) Combined with Cytosine Arabinoside and Etoposide for Treatment of Acute Leukemias" (Shiozaw, et al.).

Treatment of AML

As researchers continue to study the suspected causes, diagnosis, and treatment of AML at various medical centers, university hospitals, and other institutions, they have begun to make progress in understanding ways changes in an individual's DNA may contribute to normal bone marrow cells developing into leukemia. With an increased understanding of the genes involved in particular translocations or other chromosomal changes frequently occurring in AML, more insight into why these cells become abnormal is gained, along with the potential for the development of newer targeted therapies against AML.

The following list depicts a number of these newer target therapies, however, the newer therapies are not limited to these:

Gene Expression Profiling: This lab technique to help identify and classify different cancers utilizes microarrays to simultaneously examines the patterns of various genes in the cancer cells, rather than examining single genes.

Detection of Minimal Residual Disease: "The polymerase chain reaction (PCR) test can identify AML cells based on their gene translocations or rearrangements. This test can find one leukemia cell among a million normal cells. A PCR test can be useful in determining how completely the treatment has destroyed the AML cells" (Detailed Guide: Leukemia).

Improving Chemotherapy

Stem Cell Transplants

Targeted Therapies

New targeted drugs that specifically attack some of the genetic changes seen in AML are now being developed.

About 1 person out of 3 with AML has a mutation in the FLT3 gene. Several new drugs, called FLT3 inhibitors, target this gene. They have shown activity against AML in early studies, especially when combined with chemotherapy. So far, they are only available in clinical trials. Other gene mutations, such as changes in the c-KIT gene, also appear to be important in some cases of AML, and may become important targets for new therapies. Drugs that target this gene, such as imatinib (Gleevec) and dasatinib (Sprycel) are already used against other types of leukemia, and are now being studied against AML.

Gemtuzumab ozogamicin (Mylotarg) is a monoclonal antibody that is often used in older patients if AML doesn't respond to chemotherapy or comes back after treatment. Doctors are now studying whether this drug might be useful if given with chemotherapy earlier in the course of the disease (Detailed Guide: Leukemia).

Study I

During Study I, "Epigenetic Modification of CCAAT/Enhancer Binding Protein Expression in Acute Myeloid Leukemia," Hackanson, et al., as noted earlier, confirm C/EBP mRNA as a target for miRNA-124a. Bjorn, et al. signify that C/EBP epigenetic alterations denote a frequent event in AML and epigenetic treatment may result in down-regulation of a key hematopoietic transcription facto. Hackanson, et al. point out that the "functional loss of CCAAT/enhancer binding protein (C/EBP), a master regulatory transcription factor in the hematopoietic system, can result in a differentiation block in granulopoiesis and thus contribute to leukemic transformation. Here, we show the effect of epigenetic aberrations in regulating C/EBP expression in acute myeloid leukemia (AML)" (Abstract section, para. 1). Results from the study by Hackanson, et al. suggest that epigenetic alterations of C/EBP frequently occur in AML. In addition, epigenetic treatment result in down-regulation of a key hematopoietic transcription factor (Ibid.1).

Acute myeloid leukemia (AML) has been extensively studied at the cytogenetic, molecular, and transcriptional level. This knowledge has contributed to the subclassification of AML and translated into significant improvement of therapies (1 -- 3). Leukemic transformation to AML is a multistep process requiring the alterations of genes involved in proliferation/survival and hematopoietic differentiation (4). One such gene, CCAAT/enhancer binding protein (C/EBP), is a key transcription factor involved in the regulation of cell proliferation and differentiation in a variety of cell types, particularly in the hematopoietic system (5, 6). Whereas under physiologic conditions C/EBP is a master regulator for myeloid differentiation and granulocytic maturation, its absence results in a block of granulopoiesis, as shown in several studies (6, 7). C/EBP has gained interest in the AML field, because it has been shown that down-regulation of C/EBP protein through mutations, posttranslational modifications, and protein-protein interactions with fusion proteins AML1/ETO or CBFB-SMMHC plays a key role in leukemic transformation (6, 8 -- 11) (Hackanson, et al. Introduction section, para. 1).

Besides genetic aberrations, epigenetic modifications, such as DNA methylation and histone-tail modifications, have been shown to initiate or augment malignant transformation (12 -- 14). Global promoter studies, as well as gene-specific approaches, have revealed that aberrant promoter methylation is a common event in AML (13, 15). Because of the pharmacologic reversibility of epigenetic changes by drugs, such as the DNA-demethylating agent 5-aza-2'deoxycytidine (DAC) or the histone deacetylase (HDAC) inhibitor valproic acid, epigenetic therapy seems prominently among novel leukemia treatment strategies (16 -- 19). Whereas it is commonly seen that epigenetic treatment leads primarily to up-regulation of genes, several groups have recently shown that DNA demethylation and HDAC inhibition can also result in down-regulation of gene expression (20, 21). The molecular mechanisms underlying these findings are largely unknown, but potential mechanisms include alterations in gene expression profiles as a result of drug treatment triggering additional changes in gene expression that are independent of promoter demethylation (20) (Hackanson, et al. Introduction section, para. 2).

A third epigenetic mechanism has recently gained attention: gene expression regulation through microRNAs (miRNA). These short noncoding RNAs have been shown to down-regulate gene expression by targeting the 3' untranslated region (UTR) of their target genes. Depending on whether the specific miRNA is entirely or partially complementary to its 3' UTR binding site, down-regulation is accomplished by either mRNA degradation or translational repression, respectively (22). Besides their role in cell proliferation, differentiation, and apoptosis, recent studies have provided evidence that miRNAs are also involved in leukemogenesis (23, 24) (Hackanson, et al. Introduction section, para. 3).

Until recently, little has been known about the regulation of miRNAs, but seminal studies have now shown that hematopoietic transcription factors C/EBP and PU.1 are capable of steering miRNA-223 (miR-223) expression, which is a crucial factor in granulocytic differentiation (25, 26). Moreover, it is becoming evident that miRNAs are not only effectors of the epigenetic machinery, but they themselves can be regulated by DNA methylation (27 -- 29) (Hackanson, et al. Introduction section, para..4).

In a recent study, DNA methylation of the C/EBP core promoter was found in a small subset of AML patients and biologically linked to T-cell lineage infidelity (30). Furthermore, we have shown in two studies in lung cancer and head and neck squamous cell carcinoma that C/EBP expression is down-regulated by epigenetic mechanisms, including DNA methylation of the C/EBP upstream promoter region (31, 32). Encouraged by these results, we sought to comprehensively investigate the role of epigenetic regulation of C/EBP in AML (Hackanson, et al. Introduction section, para. 5).

Patient samples and cell lines. One hundred forty-six bone marrow samples from AML patients were obtained from the Cancer and Leukemia Group B. tissue bank and the University of Freiburg tissue bank. Bone marrow samples from seven healthy donors were collected after obtaining informed consent under a protocol approved by Ohio State University Institutional Review Board. Cell lines U937, THPI, HL60, K562, and Kasumi1 were obtained from the American Type Culture Collection, and NB4 from the German Collection of Cell Cultures. U937, THPI, and NB4 were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS); Kasumi1 was cultured in RPMI 1640 supplemented with 20% FBS; HL60 was cultured in DMEM supplemented with 10% FBS; K562 was cultured in Iscove's modified Dulbecco's medium supplemented with 10% FBS. All media were supplemented with 1% streptomycin/penicillin. CD34+ cells from a healthy donor were cultured in CellGro Medium (CellGenix) supplemented with SCF (100 ng/mL), FLT3 (100 ng/mL), IL-3 (50 ng/mL), and IL-6 (20 ng/mL). Granulocyte colony-stimulating factor (G-CSF) was given at 50 ng/mL on day 0 and day 6 (Hackanson, et al. Materials and methods section, para.1).

DAC and trichostatin a treatment. Suspension cells were seeded at a concentration of 5 x 105/mL and treated for 72 and 96 h with 200 nmol/L DAC (Sigma-Aldrich), for 24 h with 300 nmol/L trichostatin a (TSA; Sigma-Aldrich), or for 72 h with 200 nmol/L DAC followed by 24 h with 300 nmol/L TSA. Medium and drugs were replaced daily (Hackanson, et al. Materials and methods section, para. 2).

DNA and RNA isolation. Total RNA of patient samples and cell lines was isolated using Trizol (Invitrogen) following manufacturer's recommendations. Genomic DNA of patient samples and cell lines was isolated from the Trizol phase remaining after removal of the aqueous fraction containing total RNA, following manufacturer's recommendations (Hackanson, et al. Materials and methods section, para. 3).

Bisulfite treatment, bisulfite sequencing, and combined bisulfite restriction analysis assay. One microgram of genomic DNA was used for bisulfite treatment as previously described (33). The primers and PCR conditions for bisulfite sequencing and combined bisulfite restriction analysis assay (COBRA) are summarized in Supplementary Table S1 (Hackanson, et al. Materials and methods section, para. 4).

BioCOBRA. BioCOBRA was performed as recently described (34). Briefly, 20 to 40 ng of the digestion products from a regular COBRA was loaded onto a DNA 500 LabChip and assayed using the Bioanalyzer 2100. The chromatograms were visually examined; raw data were exported as CSV-files using the 2100 expert software and subsequently plotted to obtain the fluorescence values for each of the expected fragments (Hackanson, et al. Materials and methods section, para. 5).

MassARRAY. Quantitative DNA methylation analysis using the MassARRAY technique was performed by Sequenom, Inc., as previously described (35). Briefly, 1 µg of genomic DNA was treated with sodium bisulfite, PCR amplified, in vitro transcribed, and then cleaved by RNase a. The samples were then quantitatively tested for their DNA methylation status using matrix-assisted laser desorption ionization-time of flight mass spectrometry. Samples were considered methylated when the average methylation within an amplicon was >10% (Hackanson, et al. Materials and methods section, para. 6).

Real-time PCR. Total RNA (1 µg) was incubated with 2 units of DNaseI (Invitrogen) for 30 min at room temperature. The DNA-free RNA was reverse transcribed using 100 units of SuperScript II (Invitrogen) and 1 µg of oligo dT per reaction. Semiquantitative C/EBP expression was measured using SYBR Green I (Bio-Rad) in an I-Cycler (Bio-Rad). The ABL1 proto-oncogene was used as the internal control. For primer sequences, see Supplementary Table S1. 18S was used as the internal control gene as recently described (28). Experiments were done in triplicates (Hackanson, et al. Materials and methods section, para. 7).

Western blot analysis. Whole-cell lysates from cell lines were prepared by incubating 2 x 106 cells in Laemmli buffer for 10 min at 95°C. For the two AML patient samples, frozen cell pellets of bone marrow mononuclear cells, collected before and after a 10-d Decitabine treatment, were lysed with Laemmli buffer. Proteins were separated by electrophoresis on 4% to 15% gradient polyacrylamide gels (Bio-Rad) and transferred onto a nitrocellulose membrane (Hackanson, et al. Materials and methods section, para. 8).

Luciferase assay and luciferase target assay. A 634-bp fragment of C/EBP 3' UTR containing the predicted miR-124a binding site was cloned into pGL3-promoter vector (Promega) at the XbaI site, downstream of the luciferase gene according to recent descriptions (36). Using Lipofectamine 2000 (Invitrogen) K562 cells (2 x 104 per well) were then cotransfected with 0.8 µg of pGL3-C/EBP-3' UTR construct, 60 ng Renilla, and 100 nmol/L of either nontargeting RNA control oligonucleotides (Dharmacon) or miR-124a (Dharmacon) (Hackanson, et al. Materials and methods section, para. 9).

Transfection assay. HL-60 (1 x 106 per well) cells were transfected with 200 nmol/L of either a nontargeting RNA control oligonucleotides (Dharmacon) or miR-124a (Dharmacon) using Nucleofector technology (Amaxa) according to the manufacturer's instructions. At 48 h after transfection, whole-cell lysates were prepared, and Western blot for C/EBP protein was performed as described above. To check whether transfection was effective, we also transfected HL-60 cells with increasing concentrations of miR-124 (40 -- 200 nmol/L) and after 6 h washed the cell thoroughly thrice with PBS, extracted RNA, and used semiquantitative RT-PCR to detect intracellular miR-124a (Hackanson, et al. Materials and methods section, para. 10).

Statistical analysis. Normalized DNA methylation levels were compared between each cytogenetic subgroup and the normal bone marrow (NBM) group using the two-sided Wilcoxon rank sum test, and the Bonferroni procedure was used to correct for multiple testing. Differences in mRNA expression and luciferase activity, relative to the controls, were evaluated by two-sample t tests. The two-sided level of significance was set at = 0.05 (Hackanson, et al. Materials and methods section, para. 11)

DNA methylation in the upstream promoter of C/EBP. Previous studies investigated the core promoter and adjacent regions of C/EBP in AML, concluding that epigenetic silencing is a rare event in C/EBP regulation (38, 39). However, it has been shown that the upstream promoter, which also bears promoter activity, is the target of epigenetic silencing in lung and head and neck cancer (31, 32, 40). This prompted us to reevaluate the role of epigenetic silencing of C/EBP in AML. To investigate the DNA methylation patterns of C/EBP comprehensively, we conducted COBRAs and sodium bisulfite sequencing on bone marrow samples from 15 AML patients and three bone marrow samples from healthy individuals (NBM). Consistent with previous reports, we found that the core promoter [16 to -- 301 bp, relative to the transcription start site (TSS)], region 3 ( -- 918 to -- 725 bp from TSS), and region 2 ( -- 1,142 to -- 896 bp from TSS) were unmethylated (Fig. 1A -- C ). However, substantial DNA methylation was present in the upstream promoter region 1 (1,423 to -- 1,121 bp from TSS; Fig. 1B). This study covered region 1 (amplicon a), parts of the core promoter (amplicon B), as well as exon 1 (amplicon C; see Fig. 1A for location of amplicons). Whereas NBM was unmethylated, 20 of 39 AML samples (51%) and four of five leukemia cell lines (Kasumi1, ME1, NB4, U937) were methylated, with 10 samples showing methylation levels of >50% (Fig. 2A ). Aberrant DNA methylation in cell lines and patient samples was restricted to region 1 (amplicon a; Fig. 2A). Interestingly, AML samples that showed extensive methylation were derived from patients with inv (16), t (8;21), or t (15;17), thus suggesting AML subgroup-specific epigenetic alterations (Hackanson, et al. Results section, para. 1).

Aberrant DNA methylation of C/EBP is associated with cytogenetic subgroups. To quantitatively evaluate DNA methylation in a larger sample set and to confirm our observation of differential methylation in cytogenetic subgroups, we used the BioCOBRA assay and measured DNA methylation in region 1 in 94 AML samples, including 26 cases with normal karyotype, 14 cases with inv (16), 13 cases with t (8;21), 15 cases with t (15;17), 11 cases with t (9;11), and 15 cases with a complex karyotype, comprising three or more chromosomal aberrations. The DNA methylation levels in NBM that served as baseline were low (median, 0%; range, 0 -- 5%). We observed significantly higher DNA methylation levels in the inv (16) and t (15;17) cytogenetic subgroups with a median of 29% and 5%, respectively (P < 0.05; Fig. 2B). No significant differences were seen in the normal karyotype (median, 2%), t (8;21) (median, 3%), t (9;11) (median, 3%), and complex karyotype patient samples (median, 4%). However, one sample from the t (8;21) subgroup showed 54% methylation and three samples from the normal karyotype subgroup showed methylation levels of 18%, 32%, and 41%. Together these data suggest that elevated DNA methylation levels of C/EBP region 1 is associated with AML subgroups inv (16) and t (15;17); however, DNA methylation does not seem to be uniformly restricted to those two cytogenetic groups (Hackanson, et al. Results section, para. 2).

Additionally, we used an independent sample set of 52 AML patients [26 with normal karyotype, 11 with t (8;21), and 15 with t (15;17)] and applied MassARRAY technology (Fig. 2C). From the 51 evaluable samples, we detected DNA methylation in 6 of 25 patients with normal karyotype, 4 of 11 patients with t (8;21), and 13 of 15 patients with t (15;17), thus confirming the strong association of DNA methylation of C/EBP region 1 with the translocation t (15;17). It has to be mentioned that, rather being associated with cytogenetic subgroups, DNA methylation of C/EBP region 1 could also generally occur in a subset of AML patients (Hackanson, et al. Results section, para. 3).

mRNA expression of C/EBP in AML. To determine if aberrant DNA methylation in the upstream promoter affected C/EBP expression, we investigated patients with inv (16) because of their broad range of differential methylation. Using semiquantitative RT-PCR, we observed substantial differences in mRNA levels among the patients, but no correlation with DNA methylation in region 1 was seen (Fig. 3A ). The observed differences of C/EBP mRNA levels in inv (16) patients are in accordance with previous studies (8, 11, 41) (Hackanson, et al. Results section, para. 4).

Because we did not find a direct correlation between DNA methylation and C/EBP expression in AML patients, we treated leukemia cell lines (HL60, U937, NB4, K562, THPI, Kasumi1) with the DNA-demethylating agent DAC and the HDAC inhibitor TSA to determine whether expression levels change after treatment. ) (Hackanson, et al. Results section, para. 5).

The core promoter was unmethylated in all cell lines (data not shown). In contrast, U937, NB4, K562, and Kasumi1 cells, but not HL60 and THPI cells, were highly methylated in region 1 (Fig. 3B). We applied BioCOBRA and showed that DNA methylation of C/EBP region 1 in U937 decreased substantially after treatment with DAC, whereas TSA had -- as expected -- no effect on DNA methylation (Fig. 3C) (Hackanson, et al. Results section, para. 6)

As epigenetic changes are known for their transcriptional regulatory potential, we examined C/EBP mRNA levels in leukemia cell lines treated with 200 nmol/L DAC for 72 and 96 hours, 300 nmol/L TSA for 24 hours, or a combination 200 nmol/L DAC for 72 hours followed by 300 nmol/L TSA for 24 hours using semiquantitative RT-PCR (Fig. 3D). The unmethylated cell line HL60 and the methylated cell line NB4 showed significant down-regulation (P < 0.05) of C/EBP mRNA levels upon DAC and/or TSA treatment. The unmethylated cell line THPI and the methylated cell lines U937, K562, and Kasumi1 showed significant up-regulation (P < 0.05) in at least one treatment time point suggesting the involvement of epigenetic factors in the regulation of C/EBP (Fig. 3D). The heterogeneous response of these cell lines upon epigenetic treatment reflects the complexity of transcriptional regulation of C/EBP in AML with DNA methylation being only one part of the regulatory machinery. Moreover, as DAC is a globally acting substance, demethylation and reactivation of other factors, regulating C/EBP, might explain the heterogeneous response in the cell lines (Hackanson, et al. Results section, para. 7).

To further investigate the effect of DNA methylation of C/EBP region 1 and to show a potential biological meaning, we treated PCR-amplified region 1 with SSSI to methylate the DNA and cloned it in front of luciferase in the pGL3 vector. As control, we use nonmethylated region 1. Hereafter, we transfected the constructs in K562 cells and measured relative luciferase activity (Fig. 3E). We observed a significant decrease in relative luciferase activity in the cells with the methylated construct. While this is an artificial system, it provides evidence in support of our hypothesis of the biological relevance of C/EBP region 1 (Hackanson, et al. Results section, para.8).

mRNA expression and DNA methylation of C/EBP in normal hematopoiesis. As C/EBP plays a crucial role in normal hematopoiesis, we sought to investigate DNA methylation of C/EBP region 1 in CD34+ selected hematopoietic progenitors from healthy donors before and upon G-CSF stimulated granulocytic differentiation (Fig. 3F). Effective differentiation treatment was confirmed by light microscopy (data not shown). While we observed up-regulation of C/EBP mRNA during differentiation treatment (Fig. 3F, top), no substantial change of DNA methylation could be detected (Fig. 3F, bottom). This indicates that in normal hematopoiesis, DNA methylation of C/EBP region 1 plays no significant role in regulating C/EBP expression in a transcriptional manner. (Hackanson, et al. Results section, para.9)

Protein expression of C/EBP. To examine the translational consequence of demethylating and HDACi treatment, we next measured C/EBP protein expression in leukemia cell lines after treatment with DAC and TSA. Whereas the unmethylated cell lines HL60 and THPI showed high C/EBP expression, methylated cell lines NB4 and U937 showed modest expression and Kasumi1 and K562 did not express C/EBP protein (Fig. 4A ). Surprisingly, after treatment with DAC and TSA, we observed a substantial down-regulation of C/EBP protein in U937, HL60, and THPI cells. Because is has been well established that C/EBP down-regulation in Kasumi1 or K562 cells is achieved through a protein-protein interaction with AML1/ETO and BCR/ABL fusion proteins, respectively, a translational up-regulation upon epigenetic treatment would have been unexpected (8). However, the down-regulation of C/EBP protein in U937, HL60, and THPI cells could not be explained by this mechanism and therefore was intriguing (Fig. 4A) (Hackanson, et al. Results section, para. 10).

Consistent with a previous study, which indicated that the cyclin-dependent kinase inhibitor p21 could be up-regulated in AML cell lines through demethylating treatment, we observed up-regulation of p21 protein after demethylating treatment in THPI cells, supporting that our observations were unlikely a technical issue (Fig. 4B; ref. 21) (Hackanson, et al. Results section, para. 11).

Furthermore, we investigated whether demethylating treatment in vivo could also result in down-regulation of C/EBP protein. We tested unselected bone marrow cells from two AML patients before and after a 10-day treatment with 20 mg/m2/d decitabine (42). In relation to the control protein tubulin, there was no change in C/EBP protein expression, suggesting no significant effect of demethylating treatment on C/EBP protein expression in these patients (Fig. 4D) (Hackanson, et al. Results section, para. 12).

C/EBP is a target of miR-124a in vitro. The surprising finding of C/EBP protein down-regulation in leukemia cell lines after epigenetic treatment and the recent findings of epigenetic regulation of miRNAs led us to the hypothesis that a specific miRNA targeting C/EBP might explain our findings (Hackanson, et al. Results section, para. 13).

We used the publicly available TargetScan software to search for miRNAs with a potential C/EBP 3' UTR binding site. miR-124a was identified as candidate with the highest predicted likelihood to bind the C/EBP 3' UTR. Further support for C/EBP mRNA and miR-124a interaction came from a study in HeLa cells investigating putative target sequences for miR-124a (43) (Hackanson, et al. Results section, para. 14).

MiR-124a is epigenetically silenced in leukemia cells and reactivated after DAC treatment. Interestingly, the miR-124a-1 and miR-124a-3 genes but, not miR-124a-2, are located within CpG islands. To investigate whether DNA methylation is involved in miR-124a regulation, we first evaluated the DNA methylation status of the miR-124a-1 and miR-124a-3 CpG island in leukemia cell lines by COBRA and MassARRAY and found that miR-124a-3 was highly methylated in all of them but not in NBM (Fig. 5C). After treatment of cell lines with 200 nm DAC and/or 300 nm TSA, substantial DNA demethylation of miR-124a-3 was seen in all cell lines (Fig. 5D shown for U937). miR-124a-1 gene was also highly methylated in HL60 and U937 cell lines and could be demethylated by DAC treatment as shown by MassARRAY (Fig. 5E). As we were not able to detect baseline expression or up-regulation of any single pri-pre miR-124a (most likely due to technical limitations), we designed primers specific for all pre-miR-124a precursors, however, not amplifying mature miR-124a. In cell lines HL60 and U937, we detected substantial up-regulation of miR-124a precursors upon DAC treatment (Fig. 5F). Moreover, expression analysis using semiquantitative RT-PCR revealed a significant up-regulation (P < 0.05) of mature miR-124a that correlated with DNA demethylation (Fig. 5G). Taken together, these data show a significant up-regulation of miR-124a precursors and mature miR-124a upon demethylating treatment. Whether only one of the three precursors or all three are getting up-regulated could not be differentiated during this study (Hackanson, et al. Results section, para. 15).

Because it has recently been shown that Cdk6, a gene involved in cell cycle progression and a potential oncogenic factor, was down-regulated by reactivation of epigenetically silenced miR-124a in colon cancer (28), we investigated Cdk6 expression in U937, THPI, and NB4 cell lines. Cdk6 was highly expressed in U937 and NB4 cells and moderately expressed in the THPI cell line. While we saw no down-regulation in U937 and NB4 cells, CdK6 protein decreased substantially in THPI cells after demethylating treatment (Fig. 4B). Next, we transfected miR-124a in U937 cells to achieve higher intracellular miR-124a levels than upon DAC treatment. This resulted in a substantial down-regulation of Cdk6 (Fig. 4C). These finding supports our hypothesis that up-regulation of epigenetically silenced miR-124a can lead to down-regulation of two of its target genes in AML (Hackanson, et al. Results section, para. 16).

Finally, we investigated the DNA methylation status of the miR-124a-1 and miR-124a-3 genes in 52 AML samples [26 with normal karyotype, 11 with t (8;21) and 15 with t (15;17)] and in G-CSF induced and control CD 34+ hematopoietic progenitors. Independent of cytogenetic subgroups, we saw substantial methylation of miR-124a-1 and miR-124a-3 in the majority of samples (Fig. 6A and B ). Interestingly, DNA methylation of the normal control CD34+ cells was significantly lower before and upon differentiating treatment compared with the majority of AML samples, suggesting that methylation of these miR-124a genes might be an acquired event during leukemogenesis. In summary, these data indicate that miR-124a is epigenetically regulated in vitro and methylation of miR-124a-1 and miR-124a-3 is a frequent finding in AML (Hackanson, et al. Results section, para. 17).

The crucial role of the transcription factor C/EBP in lineage determination during normal hematopoiesis is well established. Reduced expression or loss of function in hematopoietic malignancies has been studied extensively, and loss of C/EBP function is thought to contribute as an early event to leukemogenesis by inhibiting myeloid differentiation (7, 44). In the present study, we investigated the epigenetic contribution to C/EBP deregulation and show that aberrant DNA methylation in the upstream promoter of C/EBP is a frequent event in AML. A distinct pattern of aberrant DNA methylation in region 1 was seen in 51% of AML patient samples, whereas the core promoter and all other investigated regions remained unmethylated. Most interesting was the finding of significantly higher DNA methylation levels in AML samples cytogenetically characterized by inv (16) and t (15;17). This AML subgroup -- specific pattern suggests a biological relevance for the aberrant DNA methylation, and the reason for this preferential methylation is focus of ongoing research. A direct correlation of DNA methylation with mRNA levels was not detectable in patient samples with inv (16), possibly due to a constitutively active core promoter. The relationship to gene expression is difficult to evaluate because we believe that epigenetic modulation of the upstream promoter is not completely abolishing expression but rather reduces expression and thus modulates the expression level. Altered gene expression levels rather than on-off switches have been reported to possess drastic effects (e.g., PU.1 expression levels; ref. 45). Therefore, we speculate that such a mechanism is operating in those AML subgroups demonstrating C/EBP methylation and cooperating with additional molecular alterations in this subgroup. Alternatively, one could speculate that epigenetic alterations are just the final marking of a gene locus that has become silenced or showed reduced expression. In this case, aberrant DNA methylation would be a biomarker for a yet unknown event occurring in specific subgroups (Hackanson, et al. Discussion section, para.1).

Interestingly, C/EBP protein levels were affected by the presence of a miRNA, miR-124a, which is also regulated by promoter methylation. This interplay of two epigenetically modulated genes offers a novel explanation for the finding of down-regulation of a key hematopoietic transcription factor after pharmacologic unmasking of methylated gene promoters (Hackanson, et al. Discussion section, para. 2).

The DNA methylation status of C/EBP in AML has been studied previously (38, 39). Both studies concluded that DNA methylation of C/EBP in AML is a rare event. However, very recently, Wouters and colleagues provided first evidence for the importance of C/EBP methylation in a small subgroup of AML (30) (Hackanson, et al. Discussion section, para. 3).

However, these studies did not examine the most upstream promoter region (region 1), in which we found aberrant DNA methylation ( -- 1,423 to -- 1,121 bp from TSS). Aberrant promoter methylation has also been described in lung cancers and head and neck cancers. Again, the core promoter was not affected by epigenetic silencing in these entities (31, 32). It is noteworthy that the DNA methylation patterns within the CpG island showed tumor-type specificity with C/EBP methylation being restricted to region 1 in head and neck cancer and AML, whereas in lung cancer also region 2 was differentially methylated. A possible explanation for this finding could be that different regulatory regions are used in different tissues, and epigenetic mechanisms interrupt the interaction of the relevant binding proteins with these regions through chromatin conformation changes. Interestingly, the sequence of the upstream methylated region ( -- 1,423 to -- 1,121 bp from TSS) is highly conserved between humans, mice, and dogs according to UCSC Genome Browser (March 2006 assembly). Also, this sequence contains two SP1 and USF binding sites, which are known transcriptional activators of C/EBP (31, 46). This mechanism has been shown for USF1/2 transcription factor binding in the C/EBP upstream promoter in lung cancer (31) (Hackanson, et al. Discussion section, para. 4).

The biological relevance of differential DNA methylation of C/EBP region 1 in AML is likely, as there are two cytogenetic subgroups, inv (16) and t (15;17), which are preferentially targeted. It has been shown that leukemia fusion proteins, such as PML/RAR [t (15;17)], are capable of recruiting DNA methyltransferases to their target genes, thereby inducing epigenetic silencing (47, 48). Therefore, one could postulate that C/EBP may be a target of PML/RAR and, moreover, that the inv (16) fusion protein CBFB-SMMHC might posses DNA methyltransferase recruiting capacity. As this offers an explanation for our observation of differential C/EBP methylation in AML, the consequence may be a selection advantage of these cells, thereby contributing to the malignant clone. In the context of recent findings that C/EBP protein is down-regulated in AML posttranslationally by fusion proteins, such as AML1/ETO [t (8;21)] or CBFB-SMMHC [inv (16)], our data support the probable collaboration of genetic and epigenetic aberrations in leukemogenesis (8, 11) (Hackanson, et al. Discussion section, para. 5).

Epigenetic silencing and activation of miRNAs after demethylating treatment have been described. These reports focused on the interaction of reactivated miRNAs with the 3' UTR of proto-oncogenes (27 -- 29). Saito and colleagues showed that epigenetically silenced miR-127 could be up-regulated by demethylating treatment and targets the proto-oncogene BCL6 (27). Lujambio and colleagues observed that the epigenetic silencing of miR-124a in colon cancer cell lines resulted in activation of cyclin D. kinase 6 (CDK6), an oncogenic factor (28). In our study we report the targeting of a candidate tumor suppressor gene (C/EBP), as well as a proto-oncogene (CDK6), by a reactivated miRNA. Especially for AML, it is intriguing to speculate that epigenetic down-regulation of miR-124a may up-regulate Cdk6, a cell cycle regulator previously shown to be associated with centrosome and numerical chromosome aberrations in AML (49) (Hackanson, et al. Discussion section, para. 6).

Finally, we want to emphasize that our findings should not be considered as negating the promising results of clinical trials with epigenetic drugs in patients with hematopoietic malignancies, especially because we do not see down-regulation of C/EBP in DAC-treated patients (16, 17). On the contrary, our data might help explain why some patients respond very well to epigenetic therapy while others do not. Moreover, it should be highlighted that a large number of genetic pathways are likely to be affected by these systemic therapies, thereby making the effect of their deregulation difficult to predict. Although we show that miR-124a methylation is common in AML patients, functional consequences, especially in patients undergoing epigenetic treatment, require careful and detailed investigations in future studies. Altogether, our data suggest that examining the aberrant epigenetic profile, including C/EBP and miR-124a, in patients before treatment might prove to be an important predictor for effectiveness of epigenetic therapy (Hackanson, et al. Conclusion section, para. 1).

Study III

The researcher notes that in Study III, Shiozawa, et al. utilized an experimental design for their study, "Preclinical Studies of Vorinostat (Suberoylanilide Hydroxamic Acid) Combined with Cytosine Arabinoside and Etoposide for Treatment of Acute Leukemias." As noted earlier in this study, Shiozawa, et al. appraised "combining cytosine arabinoside [1-?-D-arabinofuranosylcytosine (ara-C)] and/or etoposide with vorinostat for use in the treatment of acute leukemias" (Abstract). Shiozawa, et al. ultimately proffered a preclinical rationale for phase I trials of the sequential combination of vorinostat followed by ara-C and etoposide in patients evidencing advanced or refractory leukemias.

Preclinical Studies of Vorinostat (Suberoylanilide Hydroxamic Acid) Combined with Cytosine Arabinoside and Etoposide for Treatment of Acute Leukemias

Purpose: Vorinostat [suberoylanilide hydroxamic acid (SAHA)] is a potent histone deacetylase inhibitor with promising clinical efficacy as an anticancer agent. In this preclinical study, we evaluated combining cytosine arabinoside [1-?-D-arabinofuranosylcytosine (ara-C)] and/or etoposide with vorinostat for use in the treatment of acute leukemias.

Experimental Design: Cell survival was examined in vitro in HL-60 human myeloid leukemia cells and K562 myeloid blast crisis chronic myelogenous leukemia cells, using the 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt and/or fluorescein diacetate/propidium iodide assays. Drug interactions were analyzed by the combination index method (CalcuSyn) and by a novel statistical method that we developed (SynStat). Cell cycle phase distribution was measured by flow cytometry.

Results: Cytotoxic antagonism resulted when vorinostat was combined concomitantly with ara-C; however, when vorinostat was given first followed by a drug-free interval before ara-C treatment, this sequential combination was mostly synergistic. Etoposide combined with vorinostat was additive to synergistic, and the synergism became more pronounced when etoposide was given after vorinostat. Cell cycle analyses revealed that the sequence-dependent interaction of vorinostat and ara-C or etoposide reflected the arrest of cells in G1 or G2 phase during vorinostat treatment and recovery into S. phase after removal of vorinostat.

Conclusions: These findings using two independent methods to assess drug combination effects provide a preclinical rationale for phase I trials of the sequential combination of vorinostat followed by ara-C and etoposide in patients with advanced or refractory leukemias. CalcuSyn findings were concordant with those of SynStat, validating the use of the latter in analyzing drug interactions (Shiozawa, et al. Abstract section, para. 1).

Leukemia is one of the 10 leading causes of cancer deaths in the United States (1); survival of adults with relapsed and/or refractory leukemias remains unsatisfactory. Although a few patients may achieve durable survival with chemotherapy or allogeneic bone marrow transplantation, the majority will not have the latter option available due to older age, absence of a donor, or refractory/progressive disease. Salvage therapy with regimens such as cytosine arabinoside [1-?-D-arabinofuranosylcytosine (ara-C)] and anthracyclines or ara-C and etoposide may induce remission only in 25% to 30% of these patients, but responses are usually of short duration. Thus, lack of effective therapy mandates development of new approaches to improve the outcome for adults with relapsed and refractory leukemias (Shiozawa, et al. Abstract section, para. 2).

Vorinostat [suberoylanilide hydroxamic acid (SAHA)] is a second-generation hydroxamic acid -- based hybrid polar compound originally shown to be a potent inducer of differentiation in murine erythroleukemia cells at micromolar concentrations (2, 3). Subsequently, vorinostat was shown to be a strong inhibitor of histone deacetylase with direct binding to the catalytic site (2, 4) and, hence, entered a new class of anticancer agents targeted at the aberrant activity of histone deacetylase implicated in a variety of cancers (5, 6). Vorinostat is currently Food and Drug Administration approved for the treatment of refractory cutaneous T-cell lymphomas. In view of its effects on growth arrest, differentiation, and/or apoptotic cell death documented in a wide range of transformed cells in vitro and in animals (6 -- 12), vorinostat has become a focus of interest as a promising anticancer agent. Phase I/II trials have recently evaluated vorinostat as a single agent given either i.v. Or orally to patients with diverse malignancies, including leukemias. These trials indicate that vorinostat had favorable oral bioavailability compared with other histone deacetylase inhibitors, had acceptable toxicity in heavily pretreated patients, and produced objective evidence of tumor regression (13 -- 15).

These findings raise the possibility of combining vorinostat with antileukemia agents because daunorubicin, idarubicin, and etoposide have significant therapeutic activity in acute myelogenous leukemia. However, to date, there are no published preclinical studies of the combination of vorinostat with ara-C, one of the most active agents for the treatment of acute myelogenous leukemia. One approach to increase the therapeutic efficacy of salvage therapy for leukemia is to combine ara-C and etoposide with novel agents such as vorinostat that may sensitize the cells to the cytotoxic activity of the salvage drugs (Shiozawa, et al. Abstract section, para. 3).

A caution to this approach, however, is the possibility that the S-phase -- blocking effect of vorinostat may antagonize the S-phase -- dependent cytotoxicity of ara-C. Hence, in this preclinical study, we evaluated the cytotoxic interaction of vorinostat with ara-C or etoposide in human leukemia cell lines (HL-60 and K562). The cytotoxic interactions of concomitant or sequential administration of vorinostat with ara-C or etoposide were analyzed by the combination index (CalcuSyn; refs. 23, 24) and by the maximal power design (SynStat; refs. 25, 26) methods. The combination index method derives a specific parametric model by fixing the mixture ratio, whereas the maximal power design method is able to evaluate combinations across several concentration ratios. Additionally, because the cytotoxicity of ara-C and etoposide is affected by cell cycle phase (27 -- 29), we investigated the effects of alterations in cell cycle phase distribution caused by vorinostat on the sensitivity of HL-60 or K562 cells to these antileukemia drugs (Shiozawa, et al. Abstract section, para. 4).

Cell cultures

HL-60 cells were obtained from the American Type Culture Collection. K562 cells were generously provided by Dr. Yoshikazu Sugimoto of the Japanese Foundation for Cancer Research (Tokyo, Japan). Cells were cultured in RPMI 1640 (Biofluids) supplemented with 10% heat-inactivated fetal bovine serum (Gemini Bio-Products) at 37°C in a humidified atmosphere containing 5% CO2. Cells in logarithmic growth phase between passages 5 and 20 were used for these experiments (Shiozawa, et al. Materials and methods section, para. 1).

Compounds

The National Cancer Institute kindly provided vorinostat under a collaborative agreement between Merck and Co., Inc., and the Division of Cancer Treatment and Diagnosis, National Cancer Institute. Cytarabine (ara-C; Faulding Pharmaceutical Co.) and etoposide (Ben Venue Laboratories, Inc.) were obtained from the University of Maryland Medical Center Pharmacy and were reconstituted according to the instructions of the manufacturer. Stock solutions of 10 mmol/L vorinostat were prepared in DMSO and stored at -- 20°C until use (Shiozawa, et al. Materials and methods section, para. 2).

Measurement of cell survival

Unless otherwise stipulated, the number of viable cells in culture was determined at the end of 72 h of drug (or control) exposure. Cells were seeded in 96-well plates at an initial concentration of 1 x 104/mL of culture, at a final volume of 200 µL/well, and were exposed to varying concentrations of vorinostat, ara-C, or etoposide, alone or in combination either simultaneously or sequentially with vorinostat given first followed by ara-C or etoposide. Cytotoxicity was assessed using the Cell Proliferation Kit II (Roche Applied Science), a colorimetric method for quantification of viable cells that is based on the cleavage of the tetrazolium salt 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt (XTT) to form formazan dye by metabolically active cells, according to the protocol of the manufacturer (Shiozawa, et al. Materials and methods section, para.3.

Concurrent exposure to vorinostat with ara-C or etoposide

Cells were seeded into 96-well plates as described above, then treated with serial dilutions of each drug individually or with both drugs simultaneously at a fixed ratio of doses that corresponded to 0.125, 0.25, 0.5, 1, 2, 3, and 4 times the individual IC50 values. After 72 h of exposure, growth inhibition was measured using the XTT assay (Shiozawa, et al. Materials and methods section, para.4).

Interaction analysis

CalcuSyn method. To determine synergistic, additive, or antagonistic effects of the drug combinations, two methods of interaction analysis were used. The first was the combination index method of Chou and Talalay (23, 24) (Shiozawa, et al. Materials and methods section, para.5).

SynStat method. The SynStat version 1.beta software6 is a statistics-based method that utilizes the maximal power design developed by Fang and Tan (25, 26). SynStat, written in the R. programming environment, consists of three programs: SYN.design (to identify, based on dose-effect data for each of two drugs used alone, the most informative mixtures of the two drugs to be used in an experiment to assess the effect of the drugs in combination), (Shiozawa, et al. Materials and methods section, para.6)

On completion of the drug combination experiments specified by SYN.design, the SYN.test program was used to test whether there was a significant departure from additivity (specified as a 15% difference in viability within the dose range of interest with a statistical power of 80% and a 5% significance level). When significant departures from additivity were found (i.e., P < 0.05), SYN.analysis was used to perform nonparametric regression analysis to estimate the dose-response surface, which shows the joint effects of the two-drug combinations (Shiozawa, et al. Materials and methods section, para.7).

Cell cycle analysis

Cell cycle phase distributions were estimated from cell nuclei stained with propidium iodide. Briefly, cells were exposed to concentrations of vorinostat (0.1-25 µmol/L) for the time periods indicated, washed with PBS twice, fixed with ice-cold 70% ethanol, and then stored at -- 20°C until the assay. RNA was destroyed with 0.2 mg/mL RNase a (Roche) and cell nuclei were stained with 0.05 mg/mL propidium iodide. Cell cycle phase distribution was estimated from (Shiozawa, et al. Materials and methods section, para. 8).

The word "acute" in acute myeloid leukemia, as noted at the start of this chapter, denotes the rapid progress AML makes when it is not treated. In the development of a thesis, the opposite practice proves true. Without the treatment of the necessary additions to a study, the thesis will not grow. During the next chapter, Chapter III, the researcher relates the methodology the researcher utilizes to stimulate the growth of this study.

CHAPTER III

METHODOLOGY

"Many of the terms associated with leukemia, including the name of the disease itself, are derived from the Greek language.

Breaking down the words into their Greek roots makes them easier to understand"

("Leukemia").

When one breaks down words, such as leukemia, into their Greek roots, they become easier to understand. For example, "Leuk- or leuko- means white or colorless and is used to form the words 'leukemia' and 'leukocyte'. The word "-emia' means blood and is found in the words 'leukemia' and 'anemia'" ("Leukemia" Understanding Leukemia Ling section, para. 2)

This qualitative study breaks down information presented in the three clinical case studies to address the study's research questions. According to Leedy and Ormrod (2005), researchers may choose from five differing qualitative research designs for their studies. The options Leedy and Ormrod purport nclude:

1. Case study;

2. Ethnography;

3. Phenomenological study;

4. Grounded theory study;

5. Content analysis.

The researcher chose to utilize the case study methodology for this thesis focusing on treatment of AML. For this study, the researcher extracted/adapted three research questions from the three clinical studies to serve to help guide the study process, while helping ensure the researcher maintain the primary study focus.

During the analysis chapter, Chapter IV, the researcher relates various findings, retrieved from addressing the following research questions which the researcher presented during this study's introduction.

4. Does C/EBP mRNA serve as a target for miRNA-124? (Hackanson, et al.).

5. Do standard anti-inflammatory agents modulate AML cell nuclear NF-B when administered in conjunction with induction chemotherapy? (Strair, et al.).

6. How affective is the practice of "combining cytosine arabinoside [1-?-D-arabinofuranosylcytosine (ara-C)] and/or etoposide with vorinostat for use in the treatment of acute leukemias"? (Shiozawa Abstract).

The "case" in a case study may constitute an individual, a school/organization, classroom/team, activity, program, event, or ongoing process. The following portray the three various types of cases: (a): Intrinsic: understanding specific individual or situation; (b): instrumental: studying particular case as a means to larger goals (drawing general conclusions); (c): Multiple/collective: studying multiple cases as part of overall study (Qualitative research: Approaches…). This study reflects the multiple/collective type case study. During the next chapter, the analysis chapter, the researcher relates a number of findings retrieved from breaking down the three case studies and other additional information retrieved during this study's literature review.

CHAPTER IV

ANALYSIS

"In AML, the myeloid stem cells usually develop into a type of immature white blood cell called myeloblasts (or myeloid blasts)"

(Adult Acute Myeloid)

This analysis chapter relates a number of findings the researcher retrieved from the four studies this case study investigated. The researcher utilized a variety of mental imaging techniques to determine a number of findings from the studies.

I. Introductory Paragraph:

You should briefly describe your problem (problem statement) in the opening sentence. The remainder of the paragraph should discuss the organization of chapter four.

II. Demographic description:

The next section of chapter four should describe the participants in your study (number, gender, age, grade, category, etc...). You can use tables to provide further clarification.

III. Presenting and analyzing your findings:

You can organize your findings in several different ways. They are:

3. By chronology of variables

5. Other

IV. Summary of findings:

Provide a concise summary of what you found out at the conclusion of this chapter. The summary should provide a lead or transition into chapter five where you will discuss the results.

Need to Add Graphs/Tables; Introduce Each and Elaborate in Regard to Research Questions

CHAPTER V

DISCUSSION, CONCLUSION, and RECOMMENDATIONS

"AML

starts with a change to a single cell in the bone marrow"

(Acute Myelogenous Leukemia).

Introduction

The start of AML, as the introductory quote for this ending chapter contends, begins with "a change to a single cell in the bone marrow." In a similar sense, the researcher prevention, as this final chapter's introductory quote purports, "is still under investigation," this study confirms, the researcher asserts, that markers for ovarian cancers also remain under investigation. More thoughts regarding concluding this study to introduce this chapter go here

Discussion

Study Synopsis

Study I, "Epigenetic Modification of CCAAT/Enhancer Binding Protein Expression in Acute Myeloid Leukemia," by Hackanson, et al.

Study II, "Nuclear Factor- B Modulation in Patients Undergoing Induction Chemotherapy for Acute Myelogenous Leukemia," by Strair, et al.

Study III, "Preclinical Studies of Vorinostat (Suberoylanilide Hydroxamic Acid) Combined with Cytosine Arabinoside and Etoposide for Treatment of Acute Leukemias," by Shiozawa, et al.

Conclusion

1. What was the primary objective of your research?

2. What were you expecting to find?

3. What do you think was one of the biggest strengths of your research?

4. What are the strongest and weakest points about the design of your research?

Recommendations

For future studies, the researcher

Change, the researcher asserts, as noted in the quote that introduced this final chapter, also denotes one vital process that has occurred during this study. The researcher not only witnessed, but contributed to a single word transforming into a myriad of sentences that ultimately changed into a full thesis. Hopefully, the researcher purports, in time, the change initiated the start of AML will be reversed through the efforts of studies such as the three presented in this study, as well as from the efforts of the study studying the studies.