Targeting DNA Methylation in Leukemia, Myelodysplastic Syndrome, and Lymphoma: A Potential Diagnostic, Prognostic, and Therapeutic Tool.

DNA methylation represents a crucial mechanism of epigenetic regulation in hematologic malignancies. The methylation process is controlled by specific DNA methyl transferases and other regulators, which are often affected by genetic alterations. Global hypomethylation and hypermethylation of tumor suppressor genes are associated with hematologic cancer development and progression. Several epi-drugs have been successfully implicated in the treatment of hematologic malignancies, including the hypomethylating agents (HMAs) decitabine and azacytidine. However, combinations with other treatment modalities and the discovery of new molecules are still the subject of research to increase sensitivity to anti-cancer therapies and improve patient outcomes. In this review, we summarized the main functions of DNA methylation regulators and genetic events leading to changes in methylation landscapes. We provide current knowledge about target genes with aberrant methylation levels in leukemias, myelodysplastic syndromes, and malignant lymphomas. Moreover, we provide an overview of the clinical trials, focused mainly on the combined therapy of HMAs with other treatments and its impact on adverse events, treatment efficacy, and survival rates among hematologic cancer patients. In the era of precision medicine, a transition from genes to their regulation opens up the possibility of an epigenetic-based approach as a diagnostic, prognostic, and therapeutic tool.

The Impact of the Microbiome on Resistance to Cancer Treatment with Chemotherapeutic Agents and Immunotherapy.

Understanding the mechanisms of resistance to therapy in human cancer cells has become a multifaceted limiting factor to achieving optimal cures in cancer patients. Besides genetic and epigenetic alterations, enhanced DNA damage repair activity, deregulation of cell death, overexpression of transmembrane transporters, and complex interactions within the tumor microenvironment, other mechanisms of cancer treatment resistance have been recently proposed. In this review, we will summarize the preclinical and clinical studies highlighting the critical role of the microbiome in the efficacy of cancer treatment, concerning mainly chemotherapy and immunotherapy with immune checkpoint inhibitors. In addition to involvement in drug metabolism and immune surveillance, the production of microbiota-derived metabolites might represent the link between gut/intratumoral bacteria and response to anticancer therapies. Importantly, an emerging trend of using microbiota modulation by probiotics and fecal microbiota transplantation (FMT) to overcome cancer treatment resistance will be also discussed.

Epigenetic Landscape in Pancreatic Ductal Adenocarcinoma: On the Way to Overcoming Drug Resistance?

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive solid malignancies due to the rapid rate of metastasis and high resistance to currently applied cancer therapies. The complex mechanism underlying the development and progression of PDAC includes interactions between genomic, epigenomic, and signaling pathway alterations. In this review, we summarize the current research findings on the deregulation of epigenetic mechanisms in PDAC and the influence of the epigenome on the dynamics of the gene expression changes underlying epithelial-mesenchymal transition (EMT), which is responsible for the invasive phenotype of cancer cells and, therefore, their metastatic potential. More importantly, we provide an overview of the studies that uncover potentially actionable pathways. These studies provide a scientific basis to test epigenetic drug efficacy in synergy with other anticancer therapies in future clinical trials, in order to reverse acquired therapy resistance. Thus, epigenomics has the potential to generate relevant new knowledge of both a biological and clinical impact. Moreover, the potential, hurdles, and challenges of predictive biomarker discoveries will be discussed, with a special focus on the promise of liquid biopsies.

Internalization property of intestinal bacteria in colon cancer and HIV/AIDS patients.

OBJECTIVES: Bacteria from the intestinal tract of Slovak and American HIV/AIDS patients and Slovak colon cancer patients were tested for the capacity to be internalized by cells of the HL-60 cell line as well as by normal human lymphocytes. They were anticipated to possess a specific characteristic, i.e. a vigorous ability to be internalized by HL-60 cells and human lymphocytes. This assumption was confirmed by gentamicin protection assay. RESULTS: Internalization of bacteria from HIV/AIDS patients frequently resulted in partial (patients SKM1, SKM22) or complete lysis (patients SKK1-1, SKM12) of HL-60 cells. In comparison with intramucosal bacteria isolated from patients with colorectal cancer (TSG, 883, 660, 838, 536, MZRa), their capacity to internalize HL-60 cells was found to be 15-20 times higher (USP15/7, USP1/4, USP3/3, SK725/5). Partial lysis (patients USP15/7, USP3/3 and SKM22) and complete lysis (patients USP1/4, SKK1-1/1, SKM1/6, SKM12/5) were detected also after internalization of bacteria by normal human lymphocytes. Compared to the amount of intracellular bacteria isolated from patients with HIV/AIDS, the ability of bacteria from patients with colorectal cancer to internalize normal human lymphocytes was significantly lower (10-15 times), yet still higher than that of bacteria isolated from healthy people. CONCLUSIONS: Our results present the ability of bacteria of colon cancer patients and HIV/AIDS patients to internalize HL-60 cells and normal human lymphocytes. The findings underline the potentially important function of bacteria in the induction of colorectal cancer and immunodeficiency. The particularly high detection ability of bacteria from HIV/AIDS patients to internalize normal human cells emphasizes their potentially important role in the process of AIDS.

Detection of protein homolougs with HIV-1 antigens in bacteria of positive patients - phase II.

OBJECTIVES: Human immunodeficiency virus type 1 is widely accepted as the cause of AIDS (Acquires Immunodeficiency Syndrome) but it is necessary to consider other factors, not only HIV, which may be involved in AIDS process. It is apparent that a viral reservoir persists in virtually all infected individuals receiving HAART. Reservoirs were detected in macrophages and other cells of the blood system, in which even very effective HAART was not able to eliminate the virus. Over the last period of time AIDS research has been focused on the gut and other mucosal tissue as the major site of HIV infection and CD4+ T cells loss. Intestinal bacteria and cells associated with GIT are in close vicinity and so has been raised the idea that bacteria may be involved in AIDS pathogenesis. Matherial/Methods: Bacteria and yeast isolated from a cohort of 67 Cambodian and Kenyan HIV positive children and from a cohort of 62 Slovak and American AIDS patients were analyzed for detection of expression of HIV-1 antigens p17, p24, p55, gp41 and gp120 (Abcam, UK). RESULTS: By monoclonal antibodies against HIV-1 proteins p17 and p55 was detected protein with molecular weight of 45-55 kDa. In samples of Cambodian and Kenyan HIV positive children was found 35 kDa protein using MAb against HIV-specific protein p17. By using MAbs against p24 was found protein of 55-60 kDa in Cambodian and Kenyan samples but, suprisingly, no proteins were detected in bacterial extracts of American and Slovak AIDS patients by this MAbs. Using monoclonal antibodies against HIV-1 specific protein gp41 was positive signal identified in 30-35% of samples from both cohorts of patients from Kenya and Cambodia and in 75% of samples from American and Slovak patients. The protein of about 75-85 kDa was detected by MAbs against gp120 only in protein extracts obtained from yeasts Candida sp. of Cambodian and Kenyan HIV positive children. CONCLUSION: The molecular weight of 55 kDa protein was detected by MAbs anti HIV p24, p17+p55. Its molecular weight is comparable to gag-encoded Pr55Gag precursor. Surprisingly, such proteins were not found in bacterial extract from samples of American and Slovak patients by using the MAbs against HIV-specific protein p24. The protein of about 75-85 kDa was detected only in Candida species protein extracts of Cambodian and Kenyan HIV positive children by the MAbs against gp120. In Slovak and American samples, protein reacting with MAbs anti gp120 was not found. These results suggest that there are specific differences between Slovak and American HIV positive patients bacterial proteins on one side and Cambodian and Kenyan on the other. These differences may suggest a diverse bacterial evolution in various geographical areas.

Diet-driven microbiome changes and physical activity in cancer patients.

Exploring the role of the gut microbiome in oncology is gaining more attention, mainly due to its ability to shape the immune system in cancer patients. A well-balanced microbial composition forms a symbiotic relationship with the host organism. Mounting evidence supports the potential of modifiable lifestyle factors, such as diet and physical activity, in restoring intestinal dysbiosis related to cancer development and treatment. In this Minireview, we describe the host-microbiome interplay following different dietary patterns, including a high-fat diet, fiber-rich diet, diet rich in rice and beans, Mediterranean diet, ketogenic diet, and physical activity in preclinical findings and clinical settings. According to the results, nutrition is a critical factor influencing the composition of gut microbial communities. Therefore, knowledge about the patient's nutritional status in pre-treatment and treatment becomes crucial for further management. A combination of individualized dietary habits and professional training plans might help to maintain gut homeostasis, potentially improving the response to anti-cancer therapy and the quality of life in cancer survivors. However, a deep understanding of underlying mechanisms and large clinical trials are needed to uncover clinically relevant correlations for personalized treatment approaches leading to better outcomes for cancer patients.

Analysis of bacteria from intestinal tract of FAP patients for the presence of APC-like sequences.

BACKGROUND: Familial adenomatous polyposis (FAP) is a hereditary disease induced by germ-line mutations in the tumor suppressor APC gene. These initiate the early stages of the adenoma-carcinoma sequence in familial, but also in sporadic (in 80% to 90%), colon tumorigenesis. We found the presence of APC-like sequences in bacteria of FAP patients. MATERIAL/METHODS: We analyzed bacteria isolated from FAP patients' rectal swabs. Total bacterial DNA was isolated and analyzed for detection of APC-like sequences using PCR. We also tested DNA homology rate and APC-like protein production. RESULTS: We collected blood samples and rectal swabs from patients with confirmed diagnosis of FAP. They were analyzed for presence of sections from exon 15 of the APC gene. Most positive results were found in sections located exactly in the area called the MCR (mutation cluster region), where the highest frequency of APC gene mutations were identified. By sequencing PCR products from bacteria in section F-G together with a patient's DNA sample and human APC gene, we found a more than 90% DNA homology rate. We also confirmed production of APC-like protein using Western blotting. CONCLUSIONS: Our results suggested two hypotheses. The APC-like protein might have same function as a truncated APC product, which is synthesized in most cases of mutations of APC gene in the MCR region in colorectal cancer cells. Alternatively, we can consider the possible existence of horizontal transfer of genetic information between eukaryotic and prokaryotic cells. Our study can be considered as a pilot project. For confirmation of our hypotheses, further research is needed.

Study of the APC gene function in the mouse APC+/APC1638N model.

OBJECTIVES: Familial adenomatous polyposis (FAP) is an autosomal dominant disease characterized by the presence of hundreds to thousands of benign polyps in the colon. If not removed prophylactically they represent a risk of developing malignant cancer with an almost 100% penentrance. FAP is induced by germline mutation in the APC gene. Tumorigenesis launched a second somatic mutation of APC gene allele, leading to synthesis of non-functional APC protein. One of the possibilities of cancer prevention could be an alternative gene therapy using bacteria as vectors for delivery of therapeutic protein molecules. MATERIAL AND METHODS: For this purpose mice model APC+/APC1638N with mutation in one allele murine homolog of the APC gene were used. Mice were fed orally commercial nutrition enriched with 0.5 ml PBS buffer with 5% milk containing 5×108 recombinant bacterial cells DE3plys6 bearing plasmid with cloned APC gene twice a week during 42 weeks. Afterwords mice were killed by thiopental, gastrointestinal tracts were removed, microscopically, macroscopically inspected for polyps/neoplastic lesions and immunohistochemically investigated with polyclonal rabbit antibody against APC protein. RESULTS: We have cloned full-lenght APC gene into vector for expression in bacterial cells Escherichia coli BL21(DE3) and BL21(DE3) pLysS. Expression of the APC protein, induced by IPTG, was detected in protein extracts of three bacterial clones: DE3104-11, DE3pLys5, DE3pLys6. APC protein was identified by Western blot analysis using monoclonal and polyclonal antibodies against the APC protein. Bacteria of clone DE3pLys6 were orally administered to APC+/APC1638N mice with mutations in the APC gene. All transgenic mice without therapy developed adenomatous polyps in the gastrointestinal tract. Transgenic mice treated by oral administration of bacteria expressing functional APC protein developed polyps in 33.3%. The remaining four mice 66.7% were without polyps development. CONCLUSION: Administration of APC gene expressing by bacteria to transgenic mice with mutation in APC gene leads to reduction in the number of mice developing polyps in the gastrointestinal tract. The effect of bacterially expressed APC protein in elimination of intestinal polyps or tumors has been monitored. These are our preliminary results and for possible confirmation of our hypotheses still more research is needed.

Tumor microbiome - an integral part of the tumor microenvironment.

The tumor microenvironment (TME) plays a significant role in tumor progression and cancer cell survival. Besides malignant cells and non-malignant components, including immune cells, elements of the extracellular matrix, stromal cells, and endothelial cells, the tumor microbiome is considered to be an integral part of the TME. Mounting evidence from preclinical and clinical studies evaluated the presence of tumor type-specific intratumoral bacteria. Differences in microbiome composition between cancerous tissues and benign controls suggest the importance of the microbiome-based approach. Complex host-microbiota crosstalk within the TME affects tumor cell biology via the regulation of oncogenic pathways, immune response modulation, and interaction with microbiota-derived metabolites. Significantly, the involvement of tumor-associated microbiota in cancer drug metabolism highlights the therapeutic implications. This review aims to summarize current knowledge about the emerging role of tumor microbiome in various types of solid malignancies. The clinical utility of tumor microbiome in cancer progression and treatment is also discussed. Moreover, we provide an overview of clinical trials evaluating the role of tumor microbiome in cancer patients. The research focusing on the communication between the gut and tumor microbiomes may bring new opportunities for targeting the microbiome to increase the efficacy of cancer treatment and improve patient outcomes.

Confirmation of HIV-like sequences in respiratory tract bacteria of Cambodian and Kenyan HIV-positive pediatric patients.

BACKGROUND: Bacteria and yeasts isolated from respiratory tracts of 39 Cambodian and 28 Kenyan HIV-positive children were tested for the presence of HIV-1 sequences. MATERIAL/METHODS: Bacteria and yeasts from the respiratory tract (nose, pharyngeal swabs) were isolated from 39 Cambodian and 28 Kenyan HIV-positive children. Bacterial chromosomal DNA was prepared by standard protocol and by Qiagen kit. The PCR specific for HIV sequences was carried out using HIV-1-specific primers.The analysis was performed by colony and dot-blot hybridization using HIV-1-specific primers which represent gag, pol and env genes of the virus. The sequencing of some PCR products was performed on the ABI 373 DNA Sequencer. RESULTS: The majority of microbes were characterized as Staphylococcus aureus, Klebsiella pneumoniae, and resp. Candida albicans. In some cases E. coli, Streptococcus pyogenes, Proteus mirabilis and Candida tropicalis were identified. Bacteria of 16 Cambodian (41%) and 8 Kenyan (31%) children were found to be positive in colony and dot-blot DNA hybridization. By the sequencing of PCR products synthesized on the template of patients' bacterial DNA using primers 68;69 for env HIV-1 gene, homology of greater than 90% with HIV-1 isolate HXB2 (HIVHXB2CG) was revealed. CONCLUSIONS: Bacteria and yeasts from the respiratory tract of 41% of Cambodian and 31% of Kenyan HIV-positive children bear HIV-like sequences. The role of bacteria in the HIV disease process is discussed.

Targeting the gut microbiome: An emerging trend in hematopoietic stem cell transplantation.

Mounting evidence has demonstrated the critical role of the gut microbiome in different cancer treatment modalities showing intensive crosstalk between microbiota and the host immune system. In cancer patients receiving hematopoietic stem cell transplantation (HSCT), conditioning regimens including chemotherapy, radiotherapy, and immunosuppressive therapy, as well as antimicrobial prophylaxis, result in intestinal barrier disruption and massive changes in microbiota composition. According to clinical studies, a drastic loss of microbial diversity during HSCT is associated with enhanced pro-inflammatory immune response and an increased risk of transplant-related complications such as graft-versus-host disease (GvHD) and mortality. In this review, we outline the current understanding of the role of microbiota diversity in the patient response to cancer therapies and highlight the impact of changes in the gut microbiome on clinical outcomes in post-HSCT patients. Moreover, the therapeutic implications of microbiota modulation by probiotics, prebiotics, and fecal microbiota transplantation (FMT) in hematologic cancer patients receiving HSCT are discussed.

The Emerging Role of Microbiota and Microbiome in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive malignant tumors due to the absence of biomarkers for early-stage detection and poor response to therapy. Since mounting evidence supports the role of microbiota composition in tumorigenesis and cancer treatment, the link between microbiome and PDAC has been described. In this review, we summarize the current knowledge regarding the impact of the gut and oral microbiome on the risk of PDAC development. Microenvironment-driven therapy and immune system interactions are also discussed. More importantly, we provide an overview of the clinical trials evaluating the microbiota role in the risk, prognosis, and treatment of patients suffering from PDAC and solid tumors. According to the research findings, immune tolerance might result from the microbiota-derived remodeling of pancreatic tumor microenvironment. Thus, microbiome profiling and targeting represent the potential trend to enhance antitumor immunity and improve the efficacy of PDAC treatment.

Polymorphisms in the adenomatous polyposis coli gene in Slovak families suspected of FAP.

OBJECTIVES: Polymorphism in the adenomatous polyposis coli (APC) gene was analyzed in 33 families suspected of familial adenomatous polyposis (FAP) without identified APC gene mutation. Screening of 104 members of mentioned families for polymorphism in the APC gene, was performed using single strand conformation polymorphism (SSCP) and DNA sequencing. RESULTS: Twelve different types of polymorphism were found in the cohort of the families analyzed. Nine polymorphisms were located within exon 15, one within exon 6, one within exon 11 and one within exon 13. Of the 12 polymorphisms, 11 were silent substitution and only one was responsible for the amino acid change - D1822V, which was identified in 60% of the families analyzed. CONCLUSION: The most frequently detected polymorphism D1822V is potentially associated with the risk of colorectal cancer. Three detected polymorphisms - Y486Y, T1493T and S1756S - also seem to be associated with colon cancer risk. All these polymorphisms may be used as markers for diagnosis of colorectal cancer. The importance of other detected polymorphisms remains still unclear, but their involvement is being continuously observed.

The spectrum of the APC pathogenic mutations in Slovak FAP patients.

OBJECTIVES: The adenomatous polyposis coli (APC) gene was analyzed for germline mutations in 113 familial adenomatous polyposis suspected families from all over Slovakia. Mutation screening was performed using single strand conformation polymorphism (SSCP) and DNA sequencing. RESULTS: Mutations in the APC gene were found in 39 (34.5%) Slovak families and 25 different pathogenic mutations throughout the APC gene were identified. Of these, 12 mutations were deletion, one was insertion and 12 were base substitution. CONCLUSIONS: Molecular diagnostics of Slovak FAP families revealed broad palette of mutations in crucial APC gene. The patients with identified APC gene mutations were assigned to a specific therapeutic FAP program.

Detection of HIV-1 sequences in intestinal bacteria of HIV/AIDS patients.

OBJECTIVES: Bacterial DNA isolated from the intestinal tract of 11 American and 30 Slovak HIV/AIDS patients were analyzed by colony and dot blot hybridization assay for HIV-1 specific sequences. Secondly, PCR using primers specific for the HIV-1 gag, pol and env genes for detection of HIV-1 sequences in these DNA were performed. RESULTS: Intestinal bacteria DNA of HIV/AIDS patients hybridized in colony and dot blot hybridization assay for HIV-1 specific sequences. PCR products synthesized on specific primers of HIV-1 gag (115 bp), env (142 bp), pol-env (1484) genes were found to be for more than 90% homologous to the corresponding sequence in HIV-1. CONCLUSIONS: Intestinal bacteria of HIV/AIDS patients are bearing sequences for more than 90% identical with those of HIV-1.

Probiotic Survey in Cancer Patients Treated in the Outpatient Department in a Comprehensive Cancer Center.

PURPOSE: Availability without prescription restriction, low cost, and simple oral administration allow cancer patients to use probiotics without knowledge of potential risks. We present a survey of probiotic use and the association with patient tumor characteristics in cancer patients treated at the outpatient department of the National Cancer Institute in Slovakia. PATIENTS AND METHODS: Between March and December 2014, 499 patients were asked to evaluate their overall experience with probiotics by questionnaire form, including the length and method of use relative to anticancer therapy, expectations, side-effect experiences, understanding of the possible risks, dietary supplement use, and others. The relevant data were statistically evaluated. RESULTS: The cohort consisted of 323 women (64.7%) and 176 men (35.3%); 91.6% were undergoing chemotherapy (2.6% together with radiotherapy) and 8.4% had no anticancer therapy. The prevalence of probiotic use was 28.5% and only 12 patients using probiotics (8.5%) described negative side effects. Most patients declared consideration of probiotic use based on recommendation from a physician (37.3%) or a pharmacist (14.8%). Nevertheless, up to 86.6% of patients declared no knowledge of possible risks. Statistically significant correlation was found between probiotic use and age of patients (P < .008), gender (P < .023), and taking other dietary supplements (P < .000002). CONCLUSIONS: In this prospective study, we present for the first time the prevalence, side-effect experience, and aspects that most likely influence probiotic use in cancer patients. Minimal knowledge of risks underlines the importance of an active approach by oncologists to inform patients about probiotic safety.

Testing of bacteria isolated from HIV/AIDS patients in experimental models.

OBJECTIVES: Bacteria purified from the intestinal tract of HIV/AIDS patients were tested for the capacity to be internalised by cells of the HL-60 cell line. Secondly, the bacteria have been applied to the rabbit's colon in order to test their pathogenic ability. RESULTS: The ability of the bacteria to be internalised by HL-60 cells was found to be very expressive. For a more complex biological characterisation of internalised bacteria, these were applied in 6-day intervals per rectum to NZB x CA rabbits during 8 months. The administered bacteria were detected by dot blot hybridisation using HIV-1 PCR probes in the rabbits' intestinal tract after 2 months. No histological and pathological changes were recorded in the gastrointestinal epithelial cells of rabbits. CONCLUSIONS: The applied bacteria of HIV/AIDS patients were not colonised in the rabbit's colon and disappeared after 2 months. Differences between HL-60 and rabbit model are discussed.

Age and Geographical Distribution in Families with BRCA1/BRCA2 Mutations in the Slovak Republic.

Molecular diagnostics of hereditary breast and/or ovarian cancer is mainly based on detection of BRCA1 and BRCA2 germline mutations in suspected families. The aim of the study was to determine the frequency, age and geographical distribution in 130 Slovak hereditary breast and ovarian cancer (HBOC) families diagnosed within the years 2000-2004. Mutation screening was performed by single-strand conformation polymorphism (SSCP), heteroduplex analysis (HDA) and sequencing of PCR products showing an abnormal migration pattern. Twenty of 130 (15.6%) HBOC suspected families were found to carry mutations in BRCA1 or BRCA2 genes. The glossary data from the National Cancer Registry of Slovakia (NCRS) were compared with the results from HBOC suspected kindreds. Age distribution of breast cancer onset in our study group showed the highest proportion of onset in HBC families within the 5th decade of life, while NCRS reports at least a ten year later onset. These findings confirmed that cases of breast cancer under 50 years of age can be used as one of the principal criteria to assign a family as a hereditary breast and/or ovarian cancer kindred. In contrast with unselected ovarian cancer cases, about 75% of all HOC index cases were diagnosed between 40 and 49 years of age. To study the geographical distribution of hereditary breast and/or ovarian cancer, Slovakia was divided into three parts. The distribution of HBOC suspected families approximately follows this division, with an increasing number in the western area of the country.