Multiplex immunofluorescence and digital pathology as tools to evaluate variation in MYC level related to molecular inhibition and molecular sub-group in diffuse large B-cell lymphoma

Abstract

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous group of lymphomas in terms of molecular profile and clinical outcomes, highlighting the need to identify new biomarkers to stratify patients. In this thesis, the protein expression of BCL2, PLK1, MYC and p53 was evaluated in a DLBCL-cohort using multiplex immunofluorescence (mIF) to investigate correlations between protein expression and survival outcome. QuPath was used for image analysis through segmentation with Instanseg and the training of machine learning object classifiers, allowing streamlined patient classification.

A clear co-expression tendency was observed for PLK1 and MYC. High levels of MYC, PLK1 and p53 were associated with inferior prognosis after adjusting for age, gender, international prognostic index (IPI) and status. For patients with combined high BCL2 and MYC expression, high PLK1 or p53 expression further worsened the prognosis.

The same mIF panel was used to stain three DLBCL cell lines and one MCL cell line following treatment with the BCL2 inhibitor venetoclax and the PLK1 inhibitor volasertib to evaluate the treatments effect on BCL2, PLK1, MYC and p53 expression. Clear differences in protein expression could be seen depending on the mutational status of the cell lines, with a higher initial MYC and p53 expression in cell lines with rearranged MYC or mutated TP53. High MYC expression correlated with volasertib sensitivity, while BCL2 mutations were associated with venetoclax sensitivity. The combined treatment was evaluated in one cell line, indicating that the combined treatment could improve treatment response as opposed to single treatment with any of the agents.

Popular Abstract

Expression of specific proteins can predict survival outcome and response to treatment in diffuse large B-cell lymphoma

B cell lymphomas are a type of cancer that develops from B cells, a type of white blood cell that is important for the body’s immune defense. If the B cells gets damaged, they can start dividing uncontrollably and form tumors, usually in the lymph nodes. The most common type of B cell lymphoma is diffuse large B cell lymphoma (DLBCL) which is a group of lymphomas where both the disease and the response to treatment can vary greatly between individuals. To better understand why some patients have worse outcomes, patient biopsies can be investigated to find proteins whose expression levels can give insight into how the disease will progress.

In this project four different proteins were investigated in around 500 DLBCL patients. Three of them; BCL2, MYC and p53 are previously well known markers of poor prognosis. P53 normally acts as a brake for the cell as it stops damaged cells from dividing. High levels of p53 indicates that this brake no longer works and that the protein can’t stop the cells from dividing. BCL2 helps keep cancer cells alive as it prevents the cell from dying whereas MYC can increase the growth of the cells. The fourth protein, PLK1, is less studied in DLBCL but has been found to work closely together with MYC to help the cancer cells grow even faster.

To investigate these proteins, multiplex immunofluorescence was used. In this method, each protein is stained with a molecule of a specific color. All the different colors can be imaged together using a special microscope, making it possible to assess the level of each protein in every cell. The images were used to investigate if high levels of the proteins could predict the survival outcome for the patient. It was shown that high levels of MYC was linked to a lower survival probability. Additionally, patients with high levels of both BCL2 and MYC had even worse survival when PLK1 or p53 levels also were high.

The same proteins were also studied in four cell lines treated with drugs that blocks the function of either BCL2 or PLK1. It was shown that cell lines with lower levels of MYC were more resistant to treatment with the PLK1 inhibitor and that cells that had different genetic changes effecting the gene encoding BCL2 required a higher dose of the BCL2 inhibitor before dying. For one of the cell lines, it was possible to also investigate the combined treatment using both the PLK1 and the BCL2 inhibitor, where it was shown that the combination of both inhibitors made the treatment more effective.

Together these results show that MYC, PLK1 and p53 are linked to poor prognosis in DLBCL and suggest that combined PLK1 and BCL2 inhibition could improve treatment response.

• author: Öjstedt, Emma
• supervisor: Sara Ek; Sofia Klint
• organization: Department of Immunotechnology
• course: KIMM05 20261
• year: 2026
• type: H2 - Master's Degree (Two Years)
• subject: Technology and Engineering
• language: English
• id: 9236927
• date added to LUP: 2026-06-12 15:54:23
• date last changed: 2026-06-12 15:54:23