In the field of cancer research, we are concentrating on hematologic malignancies, including acute and chronic leukemia, Non-Hodgkins and Hodgkins Lymphoma. Collectively, these malignancies have been estimated to represent to third leading cause of death by cancer in Ethiopia. In addition, we have undertaken several studies on Breast cancer, the second leading cause of cancer deaths in the country.
In the field of cancer research, much of the work at AHRI has focused on improved diagnostics and prognostics, hence our plan has been to gain research experience in modern methods of diagnosis, apply these in clinical scenarios, and wherever possible to assist in the implementation of these strategies to clinical diagnosis in clinical care centers in the country. We will thus organize the review of current activities around techniques introduced.
A. Flow Cytometry
A major emphasis has been placed on development of diagnostic capacity for leukemia immunophenotyping by flow cytometry. Although peripheral morphology together with cytogenetics—where available—has represented traditional diagnostic focus, in recent years much greater use of flow cytometry, as well as molecular biology approaches to evaluate genetic aberrations, have become important tools supplementing traditional methods for both diagnosis and prognosis. Because of AHRI’s longstanding experience in flow cytometric phenotyping of the immune system, it was a logical starting point in cancer diagnosis. As MSc projects for students, we have now completed two studies, with a third nearly complete, and these have been instrumental in establishing both the usefulness and feasibility of flow cytometry in this setting. An additional PhD student, Jemal Alemu, will confirm these studies and importantly assess the impact of flow cytometric diagnosis on clinical outcomes, therapy of which may (in part) be dependent on flow results. Having completed pilot studies in flow cytometry we have catalyzed the introduction of this diagnostic modality at Black Lion, and discussions for the introduction of flow cytometry at peripheral sites in the country such as Jimma University which now are providing therapy for leukemia patients has begun.
Capacity for Immunohistochemistry is limited in Ethiopia and increasingly demanded by pathologists for diagnosis and prognosis of solid malignancies in Ethiopia. In the NCD division we currently have one PhD project with a major focus on IHC, for the diagnosis of Hodgkin’s disease, and as well have planned two additional studies, one to address NHL and a second breast cancer.
Hodgkin’s lymphoma (HL) is a distinct type of malignancy featuring rare tumor cells of B cell origin, termed Reid Steinberg cells which comprise < 1 % of cells present in the effected lymph node. These cells occur in a background of a large heterogeneous infiltrate of reactive non-malignant cell populations. The infiltrating cells include B and T lymphocytes, plasma cells macrophages and histocytes, fibroblasts, mast cells and eosinophilic granulocytes. The composition of reactive cells defines the various subtypes of HL. While histological stains have classically been utilized, newer immunohistochemical approaches aid specific diagnoses. The goals of the current study are to evaluate Hodgkins Lymphoma from excised and fixed lymph nodes using primarily immunohistochemical and RT-PCR approaches. Thus, PhD student Makka Ali, has received training in multiple histological techniques for standard H&E based diagnosis, as well as for phenotypic immunomarker analysis, and detection of Epstein Barr Virus (EBV) surface protein and cellular EBV mRNA by in situ hybridization. The identification of EBV is particularly useful as it is a common infection thought to contribute to tumorigenesis. Future work will expand sample number and included further training at Lund to evaluate gene duplication as a mechanism of molecular overexpression of PD-1, a molecule expressed by Reid Steinberg cells which may inhibiting potentially protective cytotoxic T lymphocyte responses to the tumor.
We are planning two additional projects utilizing immunohistochemistry, for breast cancer and Non-Hodgkins lymphoma diagnosis, but the projects are not yet finalized.
C. Genetics and molecular biology
Diagnostic and prognostic tests for cancer are increasingly relying on more sophisticated molecular assays to identify genetic aberrations which may contribute to tumorigenesis as well aiding the classifying subtypes of malignancies as well as predicting therapeutic responses to different drugs.
In combination with flow cytometry, PhD student Jemal Alemu will begin to examine genetic aberrations associated with acute leukemia, utilizing RT-PCR analysis as well as fluorescent-in-situ hybridization (FISH). This will project will focus on common aberrations, such as the fusion transcript RUNX1-RUNXT1 common to acute myelogenous leukemia (AML) type M2, the fusion transcript PML-RAR common to AML M3, the fusion transcript CBFB-MYH11 common to AML M4l and NPM1 mutations common to AML M1; and among B-cell AML BCR-ABL, ETV6-RUNX1, and the ABL-BCR translocation common among adult B-cell ALL (and also the chronic myeloid leukemia, see below). Identification of these aberrancies solidifies the diagnosis, and also provides important prognostic information to clinicians assisting them in guiding appropriate therapy. Jemal project has been ethically approved, he has already begun flow phenotyping, and he will soon receive FISH training at the University of Michigan.
Chronic myelogenous leukemia is associated with the pathognomonic chromosomal translocation which brings the BCR and ABL gene together forming a fusion gene, identified as the so called “Philadelphia chromosome” by cytogenetic analysis, and leading to the overexpression of a tyrosine kinase, which is the target of a class of drugs, the prototype of which is imitinab. Imitinab therapy is remarkably successful in treating CML but eventually leads to drug resistance mutations in about 10% of cases. Molecular sequencing of the ABL-BCR fusion gene to identify drug resistance mutations is the current gold standard diagnostic modality to characterize treatment failure patients and directing therapy to alternative tyrosine kinase inhibitors. The goal of PhD student Samuel Kinde, will be to characterize levels of BCR-ABL fusion products by RT-PCR to definitively identify suspect drug resistant CML, perform sequencing to identify mutations, and develop a ligation PCR approach suitable for diagnosis of drug resistance in Ethiopia. He has received training at University of Leipzig in appropriate molecular methods, has received ethical clearance, and is currently accumulating CML samples.
Considerable attention is being given to minimally invasive blood biomarker approaches for diagnostic and prognostic value. Such approaches may be particularly valuable either for early diagnosis of many cancers such as breast cancer at a stage when treatment is potentially curable, or to guide more appropriate therapy specific for a given subtype. As a first step, we have initiated a study evaluating expression of plasma miRNA among breast cancer patients compared with healthy controls. The expression level of miR-21, which inhibit phosphatases involved in down regulation of cell signaling pathways and one of the most frequently upregulated miRNA in solid tumors, and miR-195, known to target the cell signaling raf-1 gene and promote tumor cell apoptosis and decreasing breast cancer viability were significantly higher in the serum of breast cancer patients than healthy controls. Levels of two other markers previously reported to be associated with breast cancer, miR145 and miR-130b, did not different between patients and controls in our study. These results demonstrate associations with miRNA levels and breast cancer in Ethiopia and suggest further research into miRNA in breast cancer is warranted in this setting.
D. Pharmacodynamics, pharmacokinetics and pharmacogenetics
Drug therapy is a cornerstone of modern medicine, but drug toxicities and inadequate biological effects can contribute to suboptimal outcomes. There are many reasons for this, not least of which is patient compliance and inadequate education, but it is well documented that drug metabolism is highly variable from patient to patient and can be influenced by many factors including overall organ function (such as by liver and kidneys), as well as host genetic polymorphisms of metabolizing enzymes, in particular the cytochrome P450 system. The end result is the patients may have too low or too high levels of the drug. Although such effects impact all disciplines of medicine, patient variability in administered drug levels is particularly critical for chemotherapy drugs for cancer which typically have a very narrow therapeutic range. Significant toxicities, particularly on regenerating tissue such as bone marrow or hair, result from too high a level, and suboptimal levels can contribute to cancer relapse with drug resistance. Research into the many factors which impact chemotherapy drug levels, as well as defining simple assays or approaches which can be applied to help guide clinicians in patient treatment protocols is likely to lead to improved patient outcomes. AHRI is supporting several projects along these lines.
Hematologic toxicities related to standard breast cancer chemotherapy regiments involving 5-flurouracil, doxorubicin and cyclophosphamide represent major complications and require frequent dose adjustments and delays in drug administration. Host drug metabolism involves many genes, including those of the highly polymorphic cytochrome P450 system, and allelic variants of the many such genes impacts drug levels and hence chemotherapy and toxic effects. In this study we evaluated key alleles of the CYP2B6, CYP3A5, CYP2C9, CYP2C19, CYP2J2 and POR genes. We observed associations between hematological toxicity and CYP2J2*7 (CYP2J2 gene, allele 7) and POR*28 (POR gene, allele 28), and suboptimal therapy or relative dose intensity with CYP2B6*6 (CYP2B6 gene, allele 6). These indicate that some breast cancer patients’ responses to chemotherapy regiments may be better predicted by CYP genotyping. (Study by Jemal Hussein, PhD student).
6-Mercaptopurine (6-MP) is a crucial chemotherapy drug utilized in maintenance phase therapy for acute lymphocytic leukemia (ALL). Unfortunately, its metabolites can lead substantial bone marrow toxicity. Many enzymes are involved in the metabolism of 6-MP, the most well studied being thiopurine methyltransferase (TPMT). Genetic polymorphisms in this gene lead to significant different levels in different patients, contributing to unpredictable toxicity; hence TPMT genotype and levels are routinely evaluated in western settings to guide dosing in patients undergoing 6-MP therapy. In the current study, PhD student AwolMekonnen will enroll 100 ALL patients and will assess genotypes and phenotypes of TPMT as well as other enzymes and cell membrane transporters involved in 6-MP metabolism, and correlate genotypes and levels with toxicity captured from patients’ charts.
A final project is directed towards Chronic Myelogenous Leukemia (CML) patients. As previouslymentioned, such patients are currently receiving the tyrosine kinase inhibitor imatinib, but the drug is well known to lead to resistance in about 10% of the patients. The current study, by PhD student YohannesGiorges, will focus on the pharmacodynamic, pharmacokinetic, and pharmacogenetics aspects of imatinib in these patients. Blood levels of the drug attained within individual patients, and identification of patient factors which impact such levels will be assessed. Such factors include compliance and concomitant use of other medications, cytochrome P450 polymorphisms, as well as renal and hepatic function. Pharmacokinetic properties will be related to the patient’s biological response to therapy, including recorded drug related toxicities and reductions in the imatinib target molecule, the BCR-ABL1 fusion product. The project has received ethical approval, and patient sample collection underway. (Funded by AHRI core)
II. Other NCD
Diabetes, hypertension, psychiatric illness and chronic respiratory disease are many other non-communicable diseases are gaining international attention due to their rapid increase in prevalence over the past two decades and their predicted impact on health care systems. Recent studies in Ethiopia have confirmed that about half of all mortality in the country is due to non-communicable diseases, and of which the aforementioned diseases contribute significantly.
In approaching NCD research at AHRI we have taken two approaches. The first is through a multinational CEBHA consortium involving multiple institutions throughout Africa and focusing on NCD. The second has been to explore to explore topics in multiple discussions with leading clinicians in the country to assist in defining priority areas for research.
CEBHA (Collaboration for Evidence-Based Healthcare and Public Health in Africa)is a consortium of multiple African partners, including Ethiopia, Malawi, Rwanda, S. Africa and Uganda, and funded by the German Ministry of Education and Research. The emphasis is on non-communicable diseases. There are multiple research tasks, two which AHRI is involved in: The first has an overall objective of establishing and monitoring a pilot intervention by community health care workers to screen community members for cardiovascular disease (CVD) risk factors. This in turn is divided into three subtasks: to pool existing SETPS data from multiple countries to evaluate the accuracy of risk definition by substituting cholesterol levels with body mass-index—as a means to reduce screening costs, 2) to explore community attitudes towards risk and chronic disease, in a qualitative study, and 3) to training community health care workers to execute and evaluate an initial screening of 1000 community members for CVD risk.
The second task is to evaluate co-morbidities of HIV patients including diabetes, hypertension and mental illness, and develop and evaluate improved integrated strategies to manage these problems for HIV patients. At AHRI we will only be involved in the initial evaluation of co-morbidities. PhD student Haile-Michael’s thesis will be focused on these two tasks, and include as well an independent economic analysis of the cardiovascular screening approach for Ethiopia.
A large part of the CEBHA funding is oriented towards capacity building and skill development in evidence-based medicine The capacity building and skills training activities within the grant are numerous, and stress importance of evidenced based medicine and public health, as well as integration of knowledge gained from research activities into policy.
Apart from CEBHA we have contacted multiple clinicians in multiple departments at TikurAnbessa hospital to explore potential future research projects. Over the course of several meetings we probed the priorities of diabetes experts at Black Lion from a clinical care perspective, as well as to explore potential research topics of interest to them. From a clinical care perspective, high priorities were indicated as patient education as well as screening efforts for diabetes. From a research perspective high priority was placed upon establishing a large prospective cohort of diabetes patients to 1) evaluate the prevalence and rate of complications of diabetes over time, and 2) as a source of patients for nested research studies aimed towards evaluating improvements in patient care (patient education, integrated care, improved therapy regiments). We are seeking funding for this. Many other smaller research projects were also identified of interest to them (outcomes of gestational diabetes, pathogenesis of malnutrition related diabetes, prevalence of non-alcoholic steatosis among diabetes, etc).
We have also had multiple discussions with member of the Pulmonary and Critical Care Department at TikurAnbessa and have identified among chronic respiratory disease, prevalence, risk factors and role of beta-agonists and inhaled steroids to improve outcome for wood stove related chronic obstructive pulmonary disease (COPD) as a high priority.
Podoconiosis or non-filiarlial elephantiasis is a peripheral lymphoedema associated disease thought to be related to long term exposure of bare feet to red clay soil. Recent work exploring the genetics of podoconiosis has revealed a significant association with HLA-DRB0701 along with HLA-DQA201 and HLA-DQB201, all of which are in linkage disequilibrium. This strongly implies a T-cell mediated HLA class II restricted recognition of antigen presenting cells in the disease pathogenesis. In order to further characterize immune system of such patients PhD student MikiasNegash plans on comparing phenotypic characteristics of T cell subsets as well as potential antigen presenting cells such as monocyte-lineage cells and dendritic cells in the blood and peripheral lesions of such patients. In addition, serum will be analyzed for cytokine and mRNA biomarkers to identify putative disease specific markers. Finally, attempts will be made to evaluate whether putative minerals known to be present in the soil are capable of selectively stimulating T cells from podoconiosis and not control patients. The proposal is under ethical review and reagents have been ordered.