Lymphoma is currently diagnosed using tissue biopsies and monitored during treatment with PET/CT scans. Although these methods are highly valuable, they may miss very small amounts of remaining disease or detect abnormalities that are not caused by cancer. In this thesis, we investigated whether a bloodplasma test could provide additional information about the presence and activity of lymphoma.

Tumor cells release small fragments of DNA into the bloodstream, known as circulating tumor DNA (ctDNA). By analysing ctDNA, lymphoma can be detected and monitored in a minimally invasive way. To improve the sensitivity of this approach, we developed a new analytical method called MNVista, which identifies highly specific DNA mutation patterns and is able to detect extremely small amounts of tumor DNA.

Using this technique, we studied several types of B-cell lymphoma. We demonstrated that ctDNA not only accurately reflects the genetic characteristics of the tumor but can also be more sensitive than current clinical methods for monitoring treatment response and detecting residual disease.

One of the most remarkable findings was that ctDNA could already be detected in blood samples collected years before lymphoma was diagnosed. In two-thirds of the patients studied, ctDNA was present before diagnosis, and in one case around 9 years earlier. Using our method, we showed that lymphoma can leave detectable molecular traces long before symptoms develop.

These findings contribute to the development of more accurate, less invasive, and more personalised approaches for the diagnosis and monitoring of lymphoma patients.

B-cell lymphomas represent a heterogeneous group of malignancies, with diffuse large B-cell lymphoma (DLBCL) being the most common and aggressive subtype. Aggressive B-cell lymphomas may arise de novo, through transformation from indolent lymphomas such as marginal zone lymphoma (MZL), or in specific clinical contexts such as post-transplant lymphoproliferative disorders (PTLD) and relapsed/refractory DLBCL (R/R DLBCL). This thesis investigates the biological mechanisms underlying lymphoma progression and transformation, and evaluates the utility of circulating tumor DNA (ctDNA) as a biomarker across various aggressive B-cell lymphoma subtypes.
Clinical and molecular analyses identified risk factors for MZL transformation and revealed that transformed MZL frequently acquires features of germinal center B cells. Multi-omics approaches showed only subtle genomic and transcriptomic changes during transformation. Across subtypes, ctDNA emerged as a promising non-invasive biomarker for diagnosis, prognosis, and disease monitoring. In PTLD, ctDNA profiling revealed recurrent genetic alterations and closely reflected tumor characteristics and guide treatment decisions.
In R/R DLBCL, a high ctDNA tumor fraction was associated with poor prognosis. Persistent ctDNA mutations or copy number alterations provided complementary diagnostic value when combined with PET-CT, supporting their integrated use in assessing disease progression and guiding treatment decisions.
Together, these findings underscore ctDNA as a clinically informative biomarker across aggressive B-cell lymphoma subtypes, with potential to improve risk stratification and therapeutic guidance.

The efficiency of tyrosine kinase inhibitors (TKIs) displays substantial interpatient variability in non-small cell lung cancer (NSCLC), even though patients receive the same targeted therapy. This observation raises the question on how resistance to TKI therapy arises and whether treatment resistance can be predicted. In this thesis we explored some of these challenges by studying on- and off-target resistance mutations. We determined presence of pre-existing resistant minor subclones in EGFR and ALK driven NSCLC. Using the highly sensitive digital droplet PCR approach, we analyzed low-abundant resistance-associated mutations in samples obtained prior to therapy. Minor resistant clones were detected only in a small subset of the cases, indicating that most on-target resistance mutations are treatment induced or below the detection limit. Moreover, for the ALK study we observed treatment associated resistance mutations exclusively in cases where the pre- and post-treatment sample were obtained from the same organ. Off-target resistance mechanisms contributing to failure of BRAF/MEK inhibition are not yet well characterized. We explored resistance mechanism in BRAF-mutant NSCLC using whole exome sequencing. Several post-treatment specific mutations were identified in pathways previously associated with resistance to BRAF/MEK inhibition, suggesting both known and novel mechanisms may contribute to therapeutic failure in this patient subgroup. Finally, we explored feasibility of utilizing short-term ex vivo cultures from pleural effusion-derived tumor cells for in vitro drug sensitivity testing. This strategy represents a promising option to guide personalized treatment selection, although challenges remain regarding culture success rates and cellular heterogeneity.

Lung cancer is the deadliest form of cancer worldwide and one of the most common in the Netherlands, with 14,500 new diagnoses per year. The two main types are small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), with NSCLC accounting for 85% of cases. In 60-70% of adenocarcinoma cases of NSCLC, a driver gene mutation is found—a genetic alteration that drives tumor growth. This opens up possibilities for targeted therapies that specifically address these mutations.

This dissertation focuses on lung cancer with EGFR exon 20 mutations (EGFRex20+) and ROS1 or ALK fusion genes. Part I explores treatment strategies for EGFRex20+, a rare mutation that affects the function of the EGFR protein. Research shows that the effectiveness of tyrosine kinase inhibitors (TKIs) strongly depends on the specific mutation variant. The POSITION20 study evaluates osimertinib (160 mg) in patients with metastatic NSCLC, revealing a limited clinical response. Additionally, circulating tumor DNA (ctDNA) is investigated as a tool for monitoring and resistance analysis.

Part II focuses on ROS1 and ALK fusion genes. A study on ROS1+ patients indicates that crizotinib has limited efficacy, especially for brain metastases. Experiments with ex vivo cell models explore personalized treatment options. In ALK+ NSCLC, resistance is studied using sensitive techniques such as ddPCR, suggesting that some mutations may already be present before treatment begins.

These findings highlight the need for personalized treatments and further research into resistance mechanisms in lung cancer.

Protein synthesis is an energy-intensive process, with translation initiation being a key regulatory step. This thesis focuses on the roles of three translation initiation factors in cancer with the aim to identify novel therapeutic targets.
Amplification of the chromosomal region encompassing the initiation factor 4EBP1 created a synthetic dependency on FGFR1 signaling in cancer. This indicates a therapeutic potential for FGFR1 inhibitors by effectively disrupting phosphorylation of 4EBP1. We showed that 4EBP1 plays a role in regulating genes involved in insulin signaling, glucose metabolism, and the inositol pathway, three pathways contributing to cancer progression.
Treatment of sarcomas with the 4EBP1 inhibitor CR-1-31B induced apoptosis and suppressed growth in vitro and in vivo. Ribosome profiling of cells treated with CR-1-31B identified YAP and TAZ as the critical eIF4A-dependent genes.
Inhibitors of the initiation factor eIF4A like silvestrol showed promising results in treating aggressive lymphomas. However, drug resistance arose through MDR1-mediated efflux of sivestrol. A genome-wide CRISPR/Cas9 screen revealed that activation of NRF2 reduced the efficacy of the eIF4A inhibitor. This resistance could be reverted by blocking FN3K which decreased the stability of NRF2.
Using pancreatic cancer models we showed that genes with long, structured 5’UTRs rely on eIF4A for translation. CR-1-31B treatment effectively suppressed tumor growth by disrupting KRAS-dependent translation, reducing KRAS, MYC, and ERK signaling.
In conclusion, our study demonstrates that disrupting translation initiation, particularly through 4EBP1 and eIF4A inhibition, can suppress tumor progression and this offers new therapeutic strategies for cancer.