The new, simple, and reliable method for the diagnosis of brain tumors is described. It is based on a TLC quantitative determination of 5-methylcytosine (m(5)C) in relation to its damage products of DNA from tumor tissue. Currently, there is evidence that oxidative stress through reactive oxygen species (ROS) plays an important role in the etiology and progression of several human diseases. Oxidative damage of DNA, lipids, and proteins is deleterious for the cell. m(5)C, along with other basic components of DNA, is the target for ROS, which results in the appearance of new modified nucleic acid bases. If so, m(5)C residue constitutes a mutational hotspot position, whether it occurs within a nucleotide sequence of a structural gene or a regulatory region. Here, we show the results of the analysis of 82 DNA samples taken from brain tumor tissues. DNA was isolated and hydrolyzed into nucleotides, which, after labeling with [gamma-(32)P]ATP, were separated on TLC. Chromatograms were evaluated using PhosphorImager and the amounts of 5-methyldeoxycytosine (m(5)dC) were calculated as a ratio (R) of m(5)dC to m(5)dC + deoxycytosine + deoxythymidine spot intensities. The R value could not only be a good diagnostic marker for brain tumors but also a factor differentiating low-grade and high-grade gliomas. Therefore, DNA methylation pattern might be a useful tool to give a primary diagnosis of a brain tumor or as a marker for the early detection of the relapse of the disease. This method has several advantages over those existing nowadays.
Multiplexed Ion Beam Imaging (MIBI) offers high-parameter tissue imaging that is well suited for describing complex immuno-spatial features in tissues, including the enumeration of various cell phenotypes, expression of immune checkpoint proteins, and quantitative description of spatial distributions between different populations.1 The high imaging resolution combined with the rich mass spectral information in each image allows for the quantification of up to 40 biomarkers in a single slide and enables immediate processing without the need for additional imaging rounds. Leveraging this, we outline an automated machine learning framework that enables rapid, deep phenotypic and spatial profiling of tissues at the subcellular level.
Methods
Our framework consists of five steps, linked together using Apache Airflow with Kubernetes. The five steps are: 1) Background correction, 2) Cell and region segmentation, 3) Cell classification, 4) Expression quantification, and 5) Spatial analysis. Background correction defines each channel in the staining panel by ensuring that there are no image artifacts that will interfere with subsequent steps. Segmentation is based on deep learning models that leverage multiple biomarker channels to delineate cells, including challenging ones that lack dsDNA signal in the plane of imaging. Cell classification uses deep learning models that leverage staining patterns alongside known phenotypic hierarchies to first define major cell lineages, then further divide them into specific sub phenotypes. Expression quantification calculates the expression levels for each checkpoint or inducible marker of interest and computes counts and densities of each cell phenotype. Lastly, spatial analysis allows for the quantitation of immune infiltration and various cell-to-cell and cell-to-region proximity features.
Results
We demonstrate end-to-end analysis of multiple tissue types with diverse morphology and tissue architecture. The robust algorithms adapt to a wide range of tissue background and noise and achieve a high degree of accuracy in segmentation and classification. This obviates the need for multiple iterations and parameter tuning to optimize algorithm performance. The high-quality results generated by the framework have been used to discover interesting associations and spatial patterns of immune cells in the tumor microenvironment.
Conclusions
We introduce an automated machine learning based framework for deep tissue profiling from MIBI images. The combination of pre-trained deep learning models connected through Airflow's directed acyclic graphs on a Kubernetes cluster, leads to a rapid and scalable bioinformatics solution for MIBI images that can be used to uncover novel biology.
Acknowledgements
The authors would like to acknowledge Ablimit Keskin and Murat Aksoy for their contributions to the development of the framework.
Reference
Keren L, Bosse M, Thompson S, Risom T, Vijayaragavan K, McCaffrey E, et al. Mibi-TOF: A multiplexed imaging platform relates cellular phenotypes and tissue structure. Science Advances. 2019;5(10).
Abstract 5‐methylcytosine (m5C) can be used as a sensitive marker of progress of the tumor formation induced by the oxidative damage reactions. We have analyzed the amount of m5C in DNA of patients with breast and colon cancers. Two dimensional thin layer chromatography (TLC) has been used to monitor 5‐methylcytosine level in DNA extracted from cancer tissues. The level of methylation of cytosine at C‐5 position in DNA from breast cancer patients correlates well with the malignancy of tumors. Interestingly higher amount of m5C in DNA for the breast cancer patients treated with different chemotherapeutics was observed. It suggests an activation of DNA methyltransferase as well as a genomic suppression of the DNA repair genes expression. These differences clearly reflect the health condition of patients and support the global analysis of m5C in DNA as a good marker for diagnosis of neoplasia in clinical practice.
BACKGROUND:Controlled induced hypotension guarantees less blood loss and better visibility of the surgical site. The impact of hypotension on post-operative cognitive functions is still being discussed. The objective of this study was to evaluate the effects of controlled induced hypotension on the cognitive functions of patients undergoing functional endoscopic sinus surgery (FESS). MATERIAL AND METHODS:We allocated 47 patients with a good grade of preoperative cognitive functions evaluated with the Mini-Mental State Examination to 3 groups (1 – mild hypotension, 2 – intermediate hypotension, 3 – severe hypotension) according to the degree of mean intraoperative arterial pressure compared with preoperative blood pressure. Cognitive functions were evaluated preoperatively, 6 h, and 30 h postoperatively with standardized tests: the Stroop Test, Trail Making Test (TMT), and Verbal Fluency Test (VFT). A decrease in the test results and increase in the number of mistakes made were considered an impairment of cognitive functions. RESULTS:A total of 47 patients (group 1 – mild hypotension – 15, group 2 – intermediate hypotension – 19, group 3 – severe hypotension – 13) were included in the study. A significant decrease was observed in all the 3 groups after Stroop A test 6h postoperatively but it improved 30h postoperatively, without differences between the groups. Neither a significant decrease in the test results nor an increase in the number of mistakes was noted for Stroop B tests, TMT A&B tests and VFT. CONCLUSIONS:The degree of controlled intraoperative hypotension during FESS did not influence the results of psychometric tests.
In Brief Osteopontin (OPN) plays a key role in wound healing, organ fi brosis, and neural tissue response to injury. Marked OPN immunoreactivity was noted in the epidural scar and meninges, which correlated with arachnoid adhesions and thickening of dura. Signifi cant increase in number of OPN positive dorsal root ganglions neurons and loss of spinal somatosensory-evoked potentials amplitudes were also noted.
Clinical analysis was carried out in 53 cases treated surgically because of primary vertebral column neoplasms in Department of Neurosurgery in Poznań between 1976-1995. Plasmocytoma solitaire (9 cases), angioma (5 cases), osteoblastoma (4 cases) and aneurysmal bone cysts (4 cases) were observed most frequently. Neoplasms localization was as follows: cranio-vertebral junction (4 cases), cervical segment (14 cases), thoracic (23 cases) and lumbo-sacral segments (12 cases). Severe neoplastic vertebral column destruction was disclosed in 34 (64%) patients, but complete transversal spinal syndrome was noted only in 7 (13%) cases. Surgical procedures: posterior and latero-posterior approaches were applied in most cases while anterior and lateral approaches--were rarely used. In 3 cases two stage operations were performed. Intervertebral fusion or posterior autogenous graft was necessary in 39 cases.
There are no good blood and serum biomarkers for detection, follow up, or prognosis of brain tumors. However, they are needed for more detailed tumor classification, better prognosis estimation and selection of an efficient therapeutic strategy. The aim of this study was to use the epigenetic changes in DNA of peripheral blood samples as a molecular marker to diagnose brain tumors as well as other diseases. We have applied a very precise thin-layer chromatography (TLC) analysis of the global amount of 5-methylcytosine (m5C) in DNA from brain tumors, colon and breast cancer tissues and peripheral blood samples of the same patients. The m5C level in tissue DNA from different brain tumor types, expressed as R coefficient, changes within the range of 0.2–1.6 and overlaps with R of that of blood samples. It negatively correlates with the WHO malignancy grade. The global DNA hypomethylation quantitative measure in blood, demonstrates a big potential for development of non-invasive applications for detection of a low and a high grade brain tumors. We have also used this approach to analyze patients with breast and colon cancers. In all these cases the m5C amount in DNA cancer tissue match with data of blood. This study is the first to demonstrate the potential role of global m5C content in blood DNA for early detection of brain tumors and others diseases. So, genomic DNA hypomethylation is a promising marker for prognosis of various neoplasms as well as other pathologies.
Receptor for hyaluronan (HA) mediated motility (RHAMM) has been shown to work cooperatively with CD44 to mediate tumor progression,1 but its specific roles in breast cancer are still unclear. In our previous studies, we have shown tumor cell RHAMM deletion significantly inhibits cancer progression in xenograft models and that RHAMM is heterogeneously expressed within human breast tumors.2 We propose that focal RHAMM upregulation creates an invasive niche and have identified an RHAMM-dependent signature in this niche that is associated with poor prognosis of breast cancer patients.2 Importantly, we found that Type II Interferon signaling and MHC Class I & Class II Antigen Presentation pathways are co-enriched in the RHAMM high invasive niche. This indicates that RHAMM might be involved in regulating immune responses. Herein we characterize the immune infiltrates associated with the RHAMM high invasive niche using a mass spectrometry-based proteomic imaging technique.
Methods
FFPE tissues from 5 breast cancers were analyzed by multiple ion beam imaging (MIBI). Sections were stained with 20 antibodies simultaneously, including biomarkers to define immune cell phenotypes. Fields of views (FOVs) were selected from RHAMM high regions and RHAMM low regions. Novel machine-learning-based algorithms were applied to segment the images into spatially resolved single-cell data and to classify immune cell populations in RHAMM-high and RHAMM-low regions.
Results
In triple-negative breast cancer (TNBC), the RHAMM-high tumor invasive margin (TI-high) shows a high level of intra-tumoral immune cells compared to the RHAMM-low tumor core (TC-low), wherein immune cells are largely restricted to the peri-tumoral stroma (figure 1). The RHAMM-low invasive margin (TI-low) is fibrotic and only sparsely populated by macrophages. Segmented single-cell data from our novel machine learning-based algorithms show high levels of infiltrating B cells in the TI-high region (figure 2A-B). The invasive margin overall is characterized by higher levels of cytotoxic T-cells, T-helper cells, and macrophages vs. the core (figure 2C-F). Notably, the B-cell-rich TI-high region has fewer cytotoxic T cells than the TI-low region.
Conclusions
Our data highlights potentially novel interactions between RHAMM expression/function and B-cell infiltrates in TNBC. Given the dynamic roles of B-cells in cancer immunology,3 this suggests new possible avenues for immune modulation to inhibit RHAMM-supported breast cancer progression. On-going work is confirming this linkage in additional TNBC and expanding the analysis to other molecular subtypes of breast cancer.
Acknowledgements
This work was funded in part by grants from the University of Minnesota Department of Laboratory Medicine & Pathology-Molecular Genomic Pathology Division and the Masonic Cancer Center.
References
Hamilton SR, Fard SF, Paiwand FF, Tolg C, Veiseh M, Wang C, ... Turley EA. The hyaluronan receptors CD44 and Rhamm (CD168) form complexes with ERK1, 2 that sustain high basal motility in breast cancer cells. J Biol Chem. 2007;282(22):16667-16680. Tarullo SE, He Y, Daughters C, Knutson TP, Henzler C, Price M, ... Nelson AC. Receptor for Hyaluronan-Mediated Motility (RHAMM) defines an invasive niche associated with tumor progression and predicts poor outcomes in breast cancer patients. bioRxiv. 2022. Yuen GJ, Demissie E, Pillai S. B lymphocytes and cancer: a love–hate relationship. Trends in Cancer 2016;2(12):747-757.
Ethics Approval
FFPE tumor tissues (IRB Approval Study# 1409E53504) were collected from archived pathology tissue blocks with de-identified clinical data in compliance with HIPPA regulations and institutional policies. All participants had agreed to the institution's standard consent for research participation.
Hypertension is a common disease of the cardiovascular system and one of the main causes of mortality in the world. Its etiopathogenesis and molecular mechanisms are unknown. Epigenetic changes may play a role in its development. Therefore the level of 5-methylcytosine (5mC), a well-known epigenetic marker, was analyzed in DNA from the blood of essential hypertension patients.TLC chromatographic analysis of the DNA nucleotide composition was used to determine 5mC levels in blood DNA samples from 60 patients suffering from essential hypertension (30 with stage 1 and 30 with stage 2 hypertension) and 30 control subjects.The mean levels of 5mC were 1.80 + or - 0.69 in the healthy subjects, 1.14 + or - 0.48 in all the patients with essential hypertension, 1.29 + or - 0.50 in those with stage 1, and 0.99 + or - 0.42 in those with stage 2 of hypertension. Statistically significant differences in 5mC amount in DNA were observed between the control group and the whole patient group, the control group and each subgroup of patients, and the groups of patients with stage 1 and stage 2 of hypertension. The level of 5mC in the DNA of the essential hypertension patients was independent of clinical and biochemical factors.The level of 5mC in the DNA of patients suffering from essential hypertension is lower than in healthy people and depends of the progression of hypertension.
The authors present their own experience in application of transpedicular internal stabilization of the thoracic and lumbar spine. Clinical analysis was carried out in a group of 12 patients after vertebral column and spinal trauma managed surgically in the Department of Neurosurgery in Poznań between 1.06.95 and 31.12.96. Age of patients ranged from 19 to 56 years (mean age 35.08 +/- 13.04 yrs.). The level of vertebral fracture was as follows: thoracic (2 cases), thoraco-lumbar (6 cases) and lumbar (4 cases). Three patients were completely paraplegic. All patients underwent posterior or posterolateral surgical approach. The fractured parts of bones, translocated into vertebral canal were removed and nervous structures were decompressed. Transpendicular stabilization was performed after the decompression. Improvement of neurological condition was observed in 8 patients.
