To Örebro University

OBJECTIVES: HPV-positive oropharyngeal squamous cell carcinoma (OPSCC) and head and neck carcinoma of unknown primary (HNCUP) are increasing. Despite good prognosis, recurrence rates range from 10% to 25%. Surveillance with clinical controls and imaging is not always reliable. Circulating tumour human papillomavirus DNA (ctHPV-DNA) has emerged as a potential biomarker for treatment evaluation and detection of recurrence. We aimed to investigate the correlation between ctHPV-DNA in HPV+ OPSCC/HNCUP and radiologic tumour burden. Additionally, we sought to assess whether ctHPV-DNA could serve as a tool in treatment evaluation.

DESIGN: A prospective observational cohort study.

SETTING: This multicenter study involved three otolaryngology units located in central Sweden. We utilised HPV genotype-specific assays for droplet digital PCR (ddPCR) to detect ctHPV-DNA in plasma at diagnosis and follow-up. ctHPV-DNA levels were correlated to radiological tumour burden and radiological response using the Kendall Rank correlation coefficient and the Kruskal-Wallis test.

PARTICIPANTS: Patients with HPV+ OPSCC/HNCUP undergoing definitive (chemo)radiotherapy and enrolled in the CIRCOS study. RESULTS: Out of 54 patients, 51 were eligible for analyses. At baseline, ctHPV-DNA was detectable in 88%. A majority of patients with a favourable radiological evaluation according to RECIST had a corresponding undetectable ctHPV-DNA at follow-up. The levels of ctHPV-DNA at baseline correlated with total tumour volume and nodal volume (rτ = 0.39, p < 0.01, respectively rτ = 0.26, p < 0.01).

CONCLUSION: ctHPV-DNA shows correlation with tumour burden. This study strengthens the role of ctHPV-DNA as a promising biomarker for treatment evaluation in HPV-related OPC/HNCUP. With further research on serial plasma sampling, ctHPV-DNA could complement radiological treatment evaluation in HPV+ OPSCC/HNCUP.

TRIAL REGISTRATION: NCT05904327 [ClinicalTrials.gov].

Each year, nearly 20 million people are diagnosed with cancer worldwide,and over 9.7 million die of the disease. Tumor tissue sampling is essential for diagnosis and treatment, but it poses risks and may not fully represent the tumor due to heterogeneity. Additionally, limited sample sizes can hinder comprehensive testing, affecting precision diagnostics.

Circulating biomarkers offer a non-invasive alternative, as they are easily obtained from body fluids, and reflect tumor activity. These biomarkers include DNA, RNA, vesicles, proteins, metabolites, and whole tumor cells. They hold potential for screening, diagnosis, treatment selection, monitoring, and prognosis. Currently, circulating cell-free DNA (cfDNA) is the only clinically used biomarker for treatment selection and monitoring in cases without available tumor tissue.

The main aim of this thesis was to explore the clinical use of circulating biomarkers in cancer care. Paper I investigated liquid biopsy for variant analysis in lung cancer, finding that plasma cfDNA could predict overall survival and reliably detect variants in advanced cases. Paper II explored glycosaminoglycans (GAGs) as biomarkers for lung cancer, revealing that combining cfDNA and GAG profiles improved diagnostic sensitivity. Paper III developed sensitive assays to detect HPV in plasma, correlating ctHPV-DNA levels with tumor characteristics in oropharyngeal cancer. Paper IV examined methylation patterns in cfDNA, identifying regions that could distinguish cancer from other diseases using machine learning.

Overall, this thesis demonstrates the clinical potential of circulating biomarkers for cancer diagnosis, prognosis, and monitoring, emphasizing the value of multimodal approaches in enhancing detection accuracy.

We aimed to investigate the use of free glycosaminoglycan profiles (GAGomes) and cfDNA in plasma to differentiate between lung cancer and benign lung disease, in a cohort of 113 patients initially suspected of lung cancer. GAGomes were analyzed in all samples using the MIRAM® Free Glycosaminoglycan Kit with ultra-high-performance liquid chromatography and electrospray ionization triple quadrupole mass spectrometry. In a subset of samples, cfDNA concentration and NGS-data was available. We detected two GAGome features, 0S chondroitin sulfate (CS), and 4S CS, with cancer-specific changes. Based on the observed GAGome changes, we devised a model to predict lung cancer. The model, named the GAGome score, could detect lung cancer with 41.2% sensitivity (95% CI: 9.2-54.2%) at 96.4% specificity (95% CI: 95.2-100.0%, n = 113). When we combined the GAGome score with a cfDNA-based model, the sensitivity increased from 42.6% (95% CI: 31.7-60.6%, cfDNA alone) to 70.5% (95% CI: 57.4-81.5%) at 95% specificity (95% CI: 75.1-100%, n = 74). Notably, the combined GAGome and cfDNA testing improved the sensitivity, compared to cfDNA alone, especially in ASCL stage I (55.6% vs 11.1%). Our findings show that plasma GAGome profiles can enhance cfDNA testing performance, highlighting the applicability of a multiomics approach in lung cancer diagnostics.

BACKGROUND: Human papillomavirus (HPV) has emerged as a significant contributor to cancer incidence globally, particularly in the context of oropharyngeal squamous cell carcinoma (OPSCC) and cancer of unknown primary (HNCUP). This study aimed to develop and validate droplet digital PCR (ddPCR) assays for the detection of circulating tumor HPV DNA (ctHPV-DNA) in plasma, focusing on high-risk HPV genotypes associated with these cancers.

METHODS: ddPCR assays for HPV16, 18, 33, 35, 56, and 59 were developed and tested using gBlocks, HPV cell-free DNA, fragmented tumor HPV+ DNA, and plasma samples from patients with HPV+ OPSCC (n = 110) and HNCUP (n = 9).

RESULTS: Assays demonstrated robust technical sensitivity across all tested HPV genotypes. Clinical application of the assays on a cohort of patients with HPV+ OPSCC and HNCUP revealed high sensitivity (91.6%) and wide variability in ctHPV-DNA levels. Analyses revealed correlations between ctHPV-DNA levels and TNM stage and tumor viral load. The association between ctHPV-DNA and tumor viral load persisted even after adjusting for TNM stage. At posttreatment, 72.5% of samples had reached undetectable ctHPV-DNA levels. Having detectable ctHPV-DNA posttreatment was associated with a higher ctHPV-DNA level at diagnosis and higher viral load at diagnosis.

CONCLUSION: The findings underscore the potential of ctHPV-DNA as a biomarker for monitoring HPV+ cancers and offer insights into tumor dynamics. Implementation of these assays in clinical practice could enhance no-invasive treatment monitoring and recurrence detection in HPV-associated cancers.

CLINICAL TRIALS: NCT05904327.

Microsatellite instability is characterised by gains or losses of nucleotides in short tandem repeat sequences, microsatellites, dispersed throughout the human genome. Microsatellite instability status is a molecular fingerprint for DNA mismatch repair deficiency. Clinical detection of microsatellite instability status is important for identifying inherited disease in patients with colorectal and endometrial cancer but has also a prognostic value for survival and prediction of treatment response. Lately, microsatellite instability has been used as a tumor agnostic biomarker that predicts response to immune checkpoint inhibitors. To identify microsatellite instability status clinically, PCR and immunohistochemistry have been the gold standard. On the contrary, next generation sequencing provide simultaneous accession of large number of microsatellite loci and can be combined with detection of several other biomarkers. 

The national collaboration Genome Medicine Sweden have developed a solid tumour gene panel composed of 560 cancer associated genes with integrated microsatellite instability score. Our aim was to validate the microsatellite instability status based on microsatellite instability score from GMS560 DNA panel against the clinically used methods. Extracted DNA (100 ng) from formalin fixed paraffin embedded tissue sections with various tumour cell content >10% were analysed. During target enrichment sequencing analysis, allelic distribution from 5000 microsatellite markers were calculated by MSIsensor Pro to generate an instability score. 

The cohort consisted of microsatellite instable verified colorectal cancer samples (n=20), microsatellite stable solid tumour material (n=60). Preliminary results generated a microsatellite instability score for the colorectal cancer samples with a mean of 26.5 % (CI: 23.4-29.6, range: 16.9-32.3). Microsatellite stable tumour samples had a mean microsatellite instability score of 1.5 % (CI: 0.93-2.07, range: 1-4.45). 

In conclusion, we found the microsatellite instability score from GMS560 DNA panel to be both diagnostically sensitive and specific for determining MSI status due to obvious separation in instability. 

Background: The aim of this study was to investigate the clinical value of liquid biopsy as a primary source for variant analysis in lung cancer. In addition, we sought to characterize liquid biopsy variants and to correlate mutational load to clinical data.

Methods: Circulating cell-free DNA was extracted from plasma from patients with lung cancer (n = 60) and controls with benign lung disease (n = 16). Variant analysis was performed using the AVENIO ctDNA Surveillance kit and the results were correlated to clinical and variant analysis data from tumor tissue or cytology retrieved from clinical routine diagnostics.

Results: There were significantly more variants detected in lung cancer cases compared to controls (p = 0.011), but no difference between the histological subgroups of lung cancer was found (p = 0.465). Furthermore, significantly more variants were detected in patients with stage IIIb-IV disease compared to patients with stage I-IIIa (median 7 vs 4, p = 0.017). Plasma cfDNA mutational load was significantly associated with overall survival (p = 0.010). The association persisted when adjusted for stage and ECOG performance status (HR: 3.64, 95% CI 1.37-9.67, p = 0.009). Agreement between tumor and plasma samples significantly differed with stage; patients with stage IIIb-IV disease showed agreement in 88.2% of the cases with clinically relevant variants, compared to zero cases in stage I-IIIa (p = 0.004). Furthermore, one variant in EGFR, two in KRAS, and one in BRAF were detected in plasma but not in tumor samples.

Conclusion: This study concludes that in the vast majority of advanced NSCLC patients a reliable variant analysis can be performed using liquid biopsy from plasma. Furthermore, we found that the number of variants in plasma is associated with prognosis, possibly indicating a strategy for closer follow up on this crucial patient group.

Sudden cardiac death (SCD) is a tragic and traumatic event. SCD is often associated with hereditary genetic disease and in such cases, sequencing of stored formalin fixed paraffin embedded (FFPE) tissue is often crucial in trying to find a causal genetic variant. This study was designed to compare two massive parallel sequencing assays for differences in sensitivity and precision regarding variants related to SCD in FFPE material. From eight cases of SCD where DNA from blood had been sequenced using HaloPlex, corresponding FFPE samples were collected six years later. DNA from FFPE samples were amplified using HaloPlex HS, sequenced on MiSeq, representing the first method, as well as amplified using modified Twist and sequenced on NextSeq, representing the second method. Molecular barcodes were included to distinguish artefacts from true variants. In both approaches, read coverage, uniformity and variant detection were compared using genomic DNA isolated from blood and corresponding FFPE tissue, respectively. In terms of coverage uniformity, Twist performed better than HaloPlex HS for FFPE samples. Despite higher overall coverage, amplicon-based HaloPlex technologies, both for blood and FFPE tissue, suffered from design and/or performance issues resulting in genes lacking complete coverage. Although Twist had considerably lower overall mean coverage, high uniformity resulted in equal or higher fraction of genes covered at ≥ 20X. By comparing variants found in the matched samples in a pre-defined cardiodiagnostic gene panel, HaloPlex HS for FFPE material resulted in high sensitivity, 98.0% (range 96.6-100%), and high precision, 99.9% (range 99.5-100%) for moderately fragmented samples, but suffered from reduced sensitivity (range 74.2-91.1%) in more severely fragmented samples due to lack of coverage. Twist had high sensitivity, 97.8% (range 96.8-98.7%) and high precision, 99.9% (range 99.3-100%) in all analyzed samples, including the severely fragmented samples.

Background Circulating biomarkers for cancer have great potential for diagnosis as well as follow up of treatment. MicroRNAs (miRNA) are involved in the expression of a majority of proteins with different cell types having different miRNA expression. The aim of this study was to create a circulating miRNA-based model to discriminate patients with lung cancer from patients with benign lung disease. Methods Samples were collected from patients under investigation for lung cancer at Örebro University hospital. Patients were then divided into groups based on diagnosis, which resulted in NSCLC adenocarcinoma (n=24), NSCLC squamous cell carcinoma (n=13), SCLC (n=4) and a heterogeneous group consisting of different benign lung diseases (n=19). Healthy controls were collected separately (n=17). Circulating miRNA was processed using the extraction-free library preparation miRNA Whole Transcriptome Assay with probes for 2083 human mature miRNAs and analyzed with massive parallel sequencing. Differential expression between groups was estimated using edgeR. MiRNAs that had the highest impact on patient grouping were used in a sPLS discriminant analysis. The resulting classification model was validated using the leave-one-out method. Results The final model for comparison between patients with benign lung disease and patients with lung cancer contained 19 miRNAs. The model had an error rate of 15 % with errors distributed evenly between groups. A sub-analysis of patients with mutations in EGFR (n=5) and KRAS (n=6) was performed showing two distinct patterns in miRNA expression. Conclusion MiRNA shows promise as a circulating biomarker for lung cancer but may not be sufficient as an independent classifier. The predictive power may be improved by using several biomarkers in combination. The difference in expression between tumors with different mutations may be derived from alternate driving processes in these tumors.Sequencing results were standardized as counts per million (cpm), miRNA with cpm < 100 was filtered out and quantile normalization and log2 transformation was performed. Differential expression was analyzed using regression model (R software v. 3.5.1, package edgeR v. 3.24.1) with Benjamini-Hochberg correction. The miRNAs that, after correction, had a significant impact on sample groups were kept and analyzed with sparse partial least squares-regression. The resulting model was validated using leave-one-out method.

Human papilloma virus (HPV) is considered to be responsible for a large part of vaginal and vulvar carcinomas, and the p53 codon 72 polymorphism has been implicated in susceptibility to cancer induced by this virus, but with contradicting results. In this study, we have investigated the prognostic value of the codon 72 polymorphism by real-time PCR (qPCR) in two cohorts of vaginal (n = 66) and vulvar (n = 123) carcinomas. In vaginal carcinoma, arginine homozygous patients were significantly associated with a higher primary cure rate (p = 0.023) but also associated with a higher recurrence rate (p = 0.073), significant at distant locations (p = 0.009). No significant differences were found in overall survival rate (p = 0.499) or cancer-specific survival rate (p = 0.222). A higher frequency of arginine homozygosity was noted in HPV-positive tumors (p = 0.190) in comparison with HPV-negative tumors. In vulvar carcinoma, the genotype homozygous for arginine was significantly associated with a larger tumor size at diagnosis in the entire cohort (p = 0.015) and a lower cancer-specific survival rate (p = 0.024) compared with heterozygous (arginine/proline) in HPV-negative tumors. Our results indicate that the relation between HPV and the p53 codon 72 polymorphism is complex and the significance and mechanisms responsible for this relationship need to be further elucidated.