Informative markers in multiplex immunohistochemistry stainings of grade 4 IDH-mutant astrocytomas
Peltola, Sanni (2023)
2023
Bioteknologian ja biolääketieteen tekniikan kandidaattiohjelma - Bachelor's Programme in Biotechnology and Biomedical Engineering
Lääketieteen ja terveysteknologian tiedekunta - Faculty of Medicine and Health Technology
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Hyväksymispäivämäärä
2023-10-02
Julkaisun pysyvä osoite on
https://urn.fi/URN:NBN:fi:tuni-202308197673
https://urn.fi/URN:NBN:fi:tuni-202308197673
Tiivistelmä
IDH1-mutant astrocytomas are grade 2, 3 or 4 brain tumors typically found in adults. The grading is based on histological and molecular features of the tumor, and the prognosis is heavily dependent on the grade of the tumor with the best prognosis being associated with the lowest grade. The histological changes occurring in the tumors, tracking their progression from lower grade to a higher one, and picturing the molecular markers present is important in treatment and research, as it helps with diagnosis, therapy, and the development of new treatments.
The aim of this thesis is to inspect the informativity of tumor markers in multiplex immunohistochemical stainings when studying IDH-mutated astrocytomas that progressed from lower-grade to grade 4. The cohort of the study consisted of six patients (ages 18-35 years) which had two to three tumor samples (A, B, C) resected during the course of the disease. The regions of interest used for staining were decided with the help of a neuropathologist. Sample A was taken from a lower-grade tumor, sample B from a grade 4 tumor, and sample C either before A or between A and B. Whole genome sequencing and RNA sequencing data has been generated from the same tumors as a part of another study, and the results have been used as reference for this study.
Eight different tumor markers were used alongside DAPI, which stains the cell nuclei. IDH1 hotspot mutation specific antibody marks the tumor cells. TP53 mutation and resulting overexpression is common in cancer. CDKN2A deletion is associated with grade 4 tumors and is used as a classification when identifying tumors. NRG3 is a gene responsible for many intracellular processes, including proliferation and cell-survival. Multiplex immunohistochemistry allows for the study of multiple markers at the same time. The cells were classified as positive and negative in regard to their staining intensities, and the intensities and cell numbers were studied in a program named QuPath. Graphs of the results were made with RStudio and Microsoft Excel.
Some markers (p53, PDGFRa) proved to be more informative than others (NRG3, RB1). The mean average IDH-mut-positivity in the samples was 60%, which was expected from the tumor samples. The p53 results showed the amount of p53 going up as the grade got higher, though it is not yet proven if increased p53 expression correlates with higher grade in astrocytomas. CDKN2A staining was consistent with homozygous gene deletion in most of the patients that had the deletion. NRG3 showed increase in the number of positive cells between A and B despite the normalized counts of NRG3 RNA going lower in all tumors of the cohort, but one of the patients with a hemizygous deletion in B showed lessened intensities in B. Two patients had a PDGFRa amplification and the results correlated with it, but TG02 had a very dim A sample and a very bright B sample that stained mostly the non-tumorous cells across all markers, and TG05 did not have much tissue to make analysis from. RB1 results did not seem to correlate well with the WGS. Phosphorylated version of STAT3 cannot be studied with WGS or RNA sequencing, but its overexpression is considered to correlate with malignancy. For STAT3 the WGS and RNA data is available, but pSTAT3 was used in this study.
Overall, the stainings were mostly informative, but it is suggested to use other methods of research as well for diagnosis, such as WGS and RNA sequencing as done before this study.
The aim of this thesis is to inspect the informativity of tumor markers in multiplex immunohistochemical stainings when studying IDH-mutated astrocytomas that progressed from lower-grade to grade 4. The cohort of the study consisted of six patients (ages 18-35 years) which had two to three tumor samples (A, B, C) resected during the course of the disease. The regions of interest used for staining were decided with the help of a neuropathologist. Sample A was taken from a lower-grade tumor, sample B from a grade 4 tumor, and sample C either before A or between A and B. Whole genome sequencing and RNA sequencing data has been generated from the same tumors as a part of another study, and the results have been used as reference for this study.
Eight different tumor markers were used alongside DAPI, which stains the cell nuclei. IDH1 hotspot mutation specific antibody marks the tumor cells. TP53 mutation and resulting overexpression is common in cancer. CDKN2A deletion is associated with grade 4 tumors and is used as a classification when identifying tumors. NRG3 is a gene responsible for many intracellular processes, including proliferation and cell-survival. Multiplex immunohistochemistry allows for the study of multiple markers at the same time. The cells were classified as positive and negative in regard to their staining intensities, and the intensities and cell numbers were studied in a program named QuPath. Graphs of the results were made with RStudio and Microsoft Excel.
Some markers (p53, PDGFRa) proved to be more informative than others (NRG3, RB1). The mean average IDH-mut-positivity in the samples was 60%, which was expected from the tumor samples. The p53 results showed the amount of p53 going up as the grade got higher, though it is not yet proven if increased p53 expression correlates with higher grade in astrocytomas. CDKN2A staining was consistent with homozygous gene deletion in most of the patients that had the deletion. NRG3 showed increase in the number of positive cells between A and B despite the normalized counts of NRG3 RNA going lower in all tumors of the cohort, but one of the patients with a hemizygous deletion in B showed lessened intensities in B. Two patients had a PDGFRa amplification and the results correlated with it, but TG02 had a very dim A sample and a very bright B sample that stained mostly the non-tumorous cells across all markers, and TG05 did not have much tissue to make analysis from. RB1 results did not seem to correlate well with the WGS. Phosphorylated version of STAT3 cannot be studied with WGS or RNA sequencing, but its overexpression is considered to correlate with malignancy. For STAT3 the WGS and RNA data is available, but pSTAT3 was used in this study.
Overall, the stainings were mostly informative, but it is suggested to use other methods of research as well for diagnosis, such as WGS and RNA sequencing as done before this study.