Topoisomerase 1 inhibits MYC promoter activity by inducing G-quadruplex formation.

We have investigated the function of human topoisomerase 1 (TOP1) in regulation of G-quadruplex (G4) formation in the Pu27 region of the MYC P1 promoter. Pu27 is among the best characterized G4 forming sequences in the human genome and it is well known that promoter activity is inhibited upon G4 formation in this region. We found that TOP1 downregulation stimulated transcription from a promoter with wildtype Pu27 but not if the G4 motif in Pu27 was interrupted by mutation(s). The effect was not specific to the MYC promoter and similar results were obtained for the G4 forming promoter element WT21. The other major DNA topoisomerases with relaxation activity, topoisomerases 2α and ß, on the other hand, did not affect G4 dependent promoter activity. The cellular studies were supported by in vitro investigations demonstrating a high affinity of TOP1 for wildtype Pu27 but not for mutant sequences unable to form G4. Moreover, TOP1 was able to induce G4 formation in Pu27 inserted in double stranded plasmid DNA in vitro. This is the first time TOP1 has been demonstrated capable of inducing G4 formation in double stranded DNA and of influencing G4 formation in cells.

HPV testing versus p16 immunohistochemistry in oropharyngeal squamous cell carcinoma: results from the DAHANCA 19 study.

INTRODUCTION: The prognosis after primary (chemo-)radiotherapy for oropharyngeal squamous cell carcinoma (OPSCC) is affected by Human Papillomavirus (HPV) status, with a better prognosis in HPV-positive OPSCC. HPV-status is routinely assessed by p16 immunohistochemistry (IHC), but additional HPV DNA testing is debated. Also, there are numerous HPV genotypes, which prognostic role may need clarification. The purpose of this study was: (1) to test a custom-made targeted HPV next generation sequencing (NGS) panel in OPSCC, (2) to determine correlation with p16 IHC, and (3) to assess the impact of HPV DNA testing on outcome in the prospectively randomized clinical trial DAHANCA 19. MATERIALS AND METHODS: We included 271 patients with OPSCC treated with primary (chemo-)radiotherapy in the DAHANCA 19 trial. Of these, 199 (73%) were p16-positive. HPV-status was determined by targeted HPV next generation sequencing (NGS), using a custom-made HPV genotyping panel. RESULTS: HPV was detected in 194 tumor samples. p16 IHC and NGS HPV status were concordant in 265 (98%) of 271 patients, whereas we did not detect HPV DNA in 5 p16-positive tumors. HPV16 accounted for 169 of 194 HPV-positive cases (87%). HPV genotypes 18, 31, 33, 35, and 59 were also detected.Loco-regional failure and overall survival were similar whether patients were separated by p16 IHC, or HPV DNA status (p < 0.0001 for all) and did not depend on HPV genotype (p = 0.9 and p = 0.7). CONCLUSION: In the present study, HPV DNA testing or typing in a Danish OPSCC cohort did not add additional information to p16 IHC, the most widely used and accepted prognostic indicator.

A targeted expression panel for classification, gene fusion detection and PD-L1 measurements - Can molecular profiling replace immunohistochemistry in non-small cell lung cancer?

The histological classification of non-small-cell lung cancer (NSCLC) and identification of possible therapeutic targets are important for disease management. However, as biopsies are often small, with a limited amount of tumor cells, it can be challenging to obtain enough tissue for the needed number of diagnostic immunohistochemical stains and molecular analyses. In this study, we combined a small custom designed targeted expression panel with a commercial fusion transcript assay by which we were able to perform both a histological classification (transcribing the expression of the genes encoding TTF1, Napsin A, CK5/6, and the truncated P63 isoform ΔNp63 (p40) into either adenocarcinoma or squamous cell carcinoma) and an identification of fusion genes involving ALK, RET, and ROS1. The expression panel also included the PD-L1 encoding gene, CD274, in order to evaluate the PD-L1 mRNA potential for identification of patients who will benefit from immune checkpoint inhibitor treatment. We evaluated the panel using 42 NSCLC patient samples. The molecular profiling agreed with the original immunohistochemistry (IHC)-based classification in 93% of the cases. For ten of the patients, being fusion gene positive, the fusion transcripts were detected in 100%. The molecular assessment of PD-L1 also showed agreement with the original assessment made by IHC. In conclusion, this study presents a small, targeted expression panel with the potential to perform both a molecularly based histological classification and a fusion gene identification in NSCLC patients as well as identifying PD-L1 status from a very limited amount of starting material.

Rolling Circle Enhanced Detection of Specific Restriction Endonuclease Activities in Crude Cell Extracts.

Restriction endonucleases are expressed in all bacteria investigated so far and play an essential role for the bacterial defense against viral infections. Besides their important biological role, restriction endonucleases are of great use for different biotechnological purposes and are indispensable for many cloning and sequencing procedures. Methods for specific detection of restriction endonuclease activities can therefore find broad use for many purposes. In the current study, we demonstrate proof-of-concept for a new principle for the detection of restriction endonuclease activities. The method is based on rolling circle amplification of circular DNA products that can only be formed upon restriction digestion of specially designed DNA substrates. By combining the activity of the target restriction endonuclease with the highly specific Cre recombinase to generate DNA circles, we demonstrate specific detection of selected restriction endonuclease activities even in crude cell extracts. This is, to our knowledge, the first example of a sensor system that allows activity measurements of restriction endonucleases in crude samples. The presented sensor system may prove valuable for future characterization of bacteria species or strains based on their expression of restriction endonucleases as well as for quantification of restriction endonuclease activities directly in extracts from recombinant cells.

SNP-based detection of allelic imbalance: A novel approach for identifying KIAA1549-BRAF fusion in pilocytic astrocytoma using DNA sequencing.

Pilocytic astrocytoma (PA) is the most common glioma subtype found in children, and it is a non-malignant tumor type. The majority of PAs is caused by an approximately 2 Mb tandem duplication within 7q34 which creates an in-frame KIAA1549-BRAF fusion gene. The kinase domain of BRAF is fused to the N-terminal of KIAA1549, whereby BRAF is constitutively activated. We here present a novel approach for identifying KIAA1549-BRAF fusion based on single nucleotide polymorphism (SNP) analysis and next generation sequencing (NGS). Highly polymorphic SNPs in the duplicated area and in adjacent areas were selected and a custom targeted amplicon based NGS panel was designed. The panel was tested on DNA extracted from formalin fixed and paraffin embedded tissue from a retrospective cohort, consisting of biopsies from patients with PA, anaplastic astrocytoma, oligodendroglioma and glioblastoma as well as two non-tumor biopsies. The panel could distinguish chromosome 7 gain from BRAF fusion and correctly identified 8/9 PA samples with KIAA1549-BRAF fusion confirmed by RNA sequencing. The one biopsy where no fusion was detected was fresh frozen and from the RNA sequencing expected to have very low tumor content. No allelic imbalance was detected in either oligodendroglioma or in the non-tumor biopsies.

A Dual-Sensor-Based Screening System for In Vitro Selection of TDP1 Inhibitors.

DNA sensors can be used as robust tools for high-throughput drug screening of small molecules with the potential to inhibit specific enzymes. As enzymes work in complex biological pathways, it is important to screen for both desired and undesired inhibitory effects. We here report a screening system utilizing specific sensors for tyrosyl-DNA phosphodiesterase 1 (TDP1) and topoisomerase 1 (TOP1) activity to screen in vitro for drugs inhibiting TDP1 without affecting TOP1. As the main function of TDP1 is repair of TOP1 cleavage-induced DNA damage, inhibition of TOP1 cleavage could thus reduce the biological effect of the TDP1 drugs. We identified three new drug candidates of the 1,5-naphthyridine and 1,2,3,4-tetrahydroquinolinylphosphine sulfide families. All three TDP1 inhibitors had no effect on TOP1 activity and acted synergistically with the TOP1 poison SN-38 to increase the amount of TOP1 cleavage-induced DNA damage. Further, they promoted cell death even with low dose SN-38, thereby establishing two new classes of TDP1 inhibitors with clinical potential. Thus, we here report a dual-sensor screening approach for in vitro selection of TDP1 drugs and three new TDP1 drug candidates that act synergistically with TOP1 poisons.

Topoisomerase I activity and sensitivity to camptothecin in breast cancer-derived cells: a comparative study.

BACKGROUND: Camptothecin (CPT) and its derivatives are currently used as second- or third-line treatment for patients with endocrine-resistant breast cancer (BC). These drugs convert nuclear enzyme DNA topoisomerase I (TOP1) to a cell poison with the potential to damage DNA by increasing the half-life of TOP1-DNA cleavage complexes (TOP1cc), ultimately resulting in cell death. In small and non-randomized trials for BC, researchers have observed extensive variation in CPT response rates, ranging from 14 to 64%. This variability may be due to the absence of reliable selective parameters for patient stratification. BC cell lines may serve as feasible models for generation of functional criteria that may be used to predict drug sensitivity for patient stratification and, thus, lead to more appropriate applications of CPT in clinical trials. However, no study published to date has included a comparison of multiple relevant parameters and CPT response across cell lines corresponding to specific BC subtypes. METHOD: We evaluated the levels and possible associations of seven parameters including the status of the TOP1 gene (i.e. amplification), TOP1 protein expression level, TOP1 activity and CPT susceptibility, activity of the tyrosyl-DNA phosphodiesterase 1 (TDP1), the cellular CPT response and the cellular growth rate across a representative panel of BC cell lines, which exemplifies three major BC subtypes: Luminal, HER2 and TNBC. RESULTS: In all BC cell lines analyzed (without regard to subtype classification), we observed a significant overall correlation between growth rate and CPT response. In cell lines derived from Luminal and HER2 subtypes, we observed a correlation between TOP1 gene copy number, TOP1 activity, and CPT response, although the data were too limited for statistical analyses. In cell lines representing Luminal and TNBC subtypes, we observed a direct correlation between TOP1 protein abundancy and levels of enzymatic activity. In all three subtypes (Luminal, HER2, and TNBC), TOP1 exhibits approximately the same susceptibility to CPT. Of the three subtypes examined, the TNBC-like cell lines exhibited the highest CPT sensitivity and were characterized by the fastest growth rate. This indicates that breast tumors belonging to the TNBC subtype, may benefit from treatment with CPT derivatives. CONCLUSION: TOP1 activity is not a marker for CPT sensitivity in breast cancer.

Characterization and radiosensitivity of HPV-related oropharyngeal squamous cell carcinoma patient-derived xenografts.

Background: Oropharyngeal squamous cell carcinomas (OPSCC) are rising rapidly in incidence due to Human Papillomavirus (HPV) and/or tobacco smoking. Prognosis is better for patients with HPV-positive disease, but may also be influenced by tobacco smoking and other factors. There is a need to individualize treatment to minimize morbidity and improve prognosis. Patient-derived xenografts (PDX) is an emerging pre-clinical research model that may more accurately reflect the human disease, and is an attractive platform to study disease biology and develop treatments and biomarkers. In this study we describe the establishment of PDX models, compare PDX tumors to the human original, and assess the suitability of this model for radiotherapy research and biomarker development. Material and methods: Tumor biopsies from 34 patients with previously untreated OPSCC were implanted in immunodeficient mice, giving rise to 12 squamous cell carcinoma PDX models (7 HPV+, 5 HPV-). Primary and PDX tumors were characterized extensively, examining histology, immunohistochemistry, cancer gene sequencing and gene expression analysis. Radiosensitivity was assessed in vivo in a growth delay assay. Results: Established PDX models maintained histological and immunohistochemical characteristics as well as HPV-status of the primary tumor. Important cancer driver gene mutations, e.g., in TP53, PIK3CA and others, were preserved. Gene expression related to cancer stem cell markers and gene expression subtype were preserved, while gene expression related to hypoxia and immune response differed. Radiosensitivity studies showed high concordance with clinical observations. Conclusion: PDX from OPSCC preserves important molecular characteristics of the human primary tumor. Radiosensitivity were in accordance with clinically observed treatment response. The PDX model is a clinically relevant surrogate model of head and neck cancer. Perspectives include increased understanding of disease biology, which could lead to development of novel treatments and biomarkers.

Interlinked DNA nano-circles for measuring topoisomerase II activity at the level of single decatenation events.

DNA nano-structures present appealing new means for monitoring different molecules. Here, we demonstrate the assembly and utilization of a surface-attached double-stranded DNA catenane composed of two intact interlinked DNA nano-circles for specific and sensitive measurements of the life essential topoisomerase II (Topo II) enzyme activity. Topo II activity was detected via the numeric release of DNA nano-circles, which were visualized at the single-molecule level in a fluorescence microscope upon isothermal amplification and fluorescence labeling. The transition of each enzymatic reaction to a micrometer sized labeled product enabled quantitative detection of Topo II activity at the single decatenation event level rendering activity measurements in extracts from as few as five cells possible. Topo II activity is a suggested predictive marker in cancer therapy and, consequently, the described highly sensitive monitoring of Topo II activity may add considerably to the toolbox of individualized medicine where decisions are based on very sparse samples.

The Effects of Dithiothreitol on DNA.

With the novel possibilities for detecting molecules of interest with extreme sensitivity also comes the risk of encountering hitherto negligible sources of error. In life science, such sources of error might be the broad variety of additives such as dithiothreitol (DTT) used to preserve enzyme stability during in vitro reactions. Using two different assays that can sense strand interruptions in double stranded DNA, we here show that DTT is able to introduce nicks in the DNA backbone. DTT was furthermore shown to facilitate the immobilization of fluorescent DNA on an NHS-ester functionalized glass surface. Such reactions may in particular impact the readout from single molecule detection studies and other ultrasensitive assays. This was highlighted by the finding that DTT markedly decreased the signal to noise ratio in a DNA sensor based assay with single molecule resolution.

Molecular characterization of irinotecan (SN-38) resistant human breast cancer cell lines.

BACKGROUND: Studies in taxane and/or anthracycline refractory metastatic breast cancer (mBC) patients have shown approximately 30% response rates to irinotecan. Hence, a significant number of patients will experience irinotecan-induced side effects without obtaining any benefit. The aim of this study was to lay the groundwork for development of predictive biomarkers for irinotecan treatment in BC. METHODS: We established BC cell lines with acquired or de novo resistance to SN-38, by exposing the human BC cell lines MCF-7 and MDA-MB-231 to either stepwise increasing concentrations over 6 months or an initial high dose of SN-38 (the active metabolite of irinotecan), respectively. The resistant cell lines were analyzed for cross-resistance to other anti-cancer drugs, global gene expression, growth rates, TOP1 and TOP2A gene copy numbers and protein expression, and inhibition of the breast cancer resistance protein (ABCG2/BCRP) drug efflux pump. RESULTS: We found that the resistant cell lines showed 7-100 fold increased resistance to SN-38 but remained sensitive to docetaxel and the non-camptothecin Top1 inhibitor LMP400. The resistant cell lines were characterized by Top1 down-regulation, changed isoelectric points of Top1 and reduced growth rates. The gene and protein expression of ABCG2/BCRP was up-regulated in the resistant sub-lines and functional assays revealed BCRP as a key mediator of SN-38 resistance. CONCLUSIONS: Based on our preclinical results, we suggest analyzing the predictive value of the BCRP in breast cancer patients scheduled for irinotecan treatment. Moreover, LMP400 should be tested in a clinical setting in breast cancer patients with resistance to irinotecan.

Optimized Detection of Plasmodium falciparum Topoisomerase I Enzyme Activity in a Complex Biological Sample by the Use of Molecular Beacons.

The so-called Rolling Circle Amplification allows for amplification of circular DNA structures in a manner that can be detected in real-time using nucleotide-based molecular beacons that unfold upon recognition of the DNA product, which is being produced during the amplification process. The unfolding of the molecular beacons results in a fluorescence increase as the Rolling Circle Amplification proceeds. This can be measured in a fluorometer. In the current study, we have investigated the possibility of using two different molecular beacons to detect two distinct Rolling Circle Amplification reactions proceeding simultaneously and in the same reaction tube by measurement of fluorescence over time. We demonstrate the application of this fluorometric readout method, for automated and specific detection of the activity of the type IB topoisomerase from the malaria parasite Plasmodium falciparum in the presence of human cell extract containing the related topoisomerase I from humans. The obtained results point towards a future use of the presented assay setup for malaria diagnostics or drug screening purposes. In longer terms the method may be applied more broadly for real-time sensing of various Rolling Circle Amplification reactions.

In-situ hybridization-based quantification of hTR: a possible biomarker in malignant melanoma.

AIMS: Telomerase is reactivated in most cancers and there is accumulating evidence that this is a driver event in malignant melanoma (MM). Thus, our aim was to evaluate if in-situ hybridization (ISH)-based quantification of telomerase RNA (hTR) could be used to distinguish MM from naevi, and if there was a correlation with the Breslow thickness. RESULTS AND METHODS: We created a tissue microarray (TMA) from formalin-fixed and paraffin-embedded tissue samples from 17 MM and 23 naevi, performed ISH targeting hTR, and quantified the signals. We found a more than eightfold greater number of hTR signals per nucleus in the MM samples compared to the naevi, and a positive correlation (P = 0.0381) between the number of hTR signals per nucleus and the Breslow thickness. CONCLUSION: Quantification of hTR ISH signals clearly distinguish MM from naevi (P < 0.0001) and the number of signals per nucleus correlates with the Breslow thickness, suggesting that hTR might be a valuable biomarker in MM. Furthermore, as ISH-based detection requires the presence of both hTR and telomerase reverse transcriptase (hTERT), it might be an indicator of active telomerase and thus have future relevance as a predictive biomarker for anti-telomerase treatment.

Correlation between topoisomerase I and tyrosyl-DNA phosphodiesterase 1 activities in non-small cell lung cancer tissue.

Topoisomerase I (TOP1) regulates DNA topology during replication and transcription whereas tyrosyl-DNA phosphodiesterase 1 (TDP1) is involved in the repair of several types of DNA damages, including damages from defective TOP1 catalysis. TOP1 is the target of chemotherapeutic drugs of the camptothecin family (CPT). TDP1 has in cell line based assays been shown to counteract the effect of CPT. We have quantified the enzymatic activities of TOP1 and TDP1 in paired (tumor and adjacent non-tumor) samples from non-small cell lung cancer (NSCLC) patients and show that in NSCLC TOP1 and TDP1 activities are significantly upregulated in the tumor tissue. Furthermore, we found a positive correlation between the TDP1 activity and the tumor percentage (TOP1 activity did not correlate with the tumor percentage) as well as between the activities of TOP1 and TDP1 both within the tumor and the non-tumor group. That TDP1 activity was upregulated in all tumor samples and correlated with the tumor percentage suggest that it must play a highly important function in NSCLC. This could be to protect against TOP1 mediated DNA damage as the activity of TOP1 likewise was upregulated in the majority of tumor samples and correlated positively to the TDP1 activity. Regardless, the finding that the TOP1 and TDP1 activities are upregulated and correlate positively suggests that combinatorial treatment targeting both activities could be advantageous in NSCLC.

Topoisomerase I as a biomarker: detection of activity at the single molecule level.

Human topoisomerase I (hTopI) is an essential cellular enzyme. The enzyme is often upregulated in cancer cells, and it is a target for chemotherapeutic drugs of the camptothecin (CPT) family. Response to CPT-based treatment is dependent on hTopI activity, and reduction in activity, and mutations in hTopI have been reported to result in CPT resistance. Therefore, hTOPI gene copy number, mRNA level, protein amount, and enzyme activity have been studied to explain differences in cellular response to CPT. We show that Rolling Circle Enhanced Enzyme Activity Detection (REEAD), allowing measurement of hTopI cleavage-religation activity at the single molecule level, may be used to detect posttranslational enzymatic differences influencing CPT response. These differences cannot be detected by analysis of hTopI gene copy number, mRNA amount, or protein amount, and only become apparent upon measuring the activity of hTopI in the presence of CPT. Furthermore, we detected differences in the activity of the repair enzyme tyrosyl-DNA phosphodiesterase 1, which is involved in repair of hTopI-induced DNA damage. Since increased TDP1 activity can reduce cellular CPT sensitivity we suggest that a combined measurement of TDP1 activity and hTopI activity in presence of CPT will be the best determinant for CPT response.

Characterization of the genomic landscape of HIV-associated lymphoma reveals heterogeneity across histological subtypes.

OBJECTIVE: Individuals with human immunodeficiency virus (HIV) experience an increased risk of lymphoma, making this an important cause of death among people with HIV. Nevertheless, little is known regarding the underlying genetic aberrations, which we therefore set out to characterize. DESIGN: We conducted next-generation panel sequencing to explore the mutational status of diagnostic lymphoma biopsies from 18 patients diagnosed with lymphoma secondary to HIV infection. METHODS: Ion Torrent next-generation sequencing was performed with an AmpliSeq panel on diagnostic lymphoma biopsies from HIV-associated B-cell lymphomas (n = 18), comprising diffuse large B-cell lymphoma (n = 9), classic Hodgkin lymphoma (n = 6), Burkitt lymphoma (n = 2), follicular lymphoma (n = 1), and marginal zone lymphoma (n = 1). The panel comprised 69 lymphoid- and/or myeloid-relevant genes, in which either the entire coding sequence or a hotspot region was sequenced. RESULTS: Among the 18 lymphomas, we detected 213 variants. The number of detected mutations ranged from 4 to 41 per tumor distributed among 42 genes, including both exonic and intronic regions. The most frequently mutated genes included KMT2D (67%), TNFAIP3 (50%), and TP53 (61%). Notably, no gene was found to harbor variants across all the HIV-associated lymphomas, nor did we find subtype-specific variants. While some variants were shared among patients, most were unique to the individual patient and were often not reported as malignant genetic variants in databases. CONCLUSION: Our findings demonstrate genetic heterogeneity across histological subtypes of HIV-associated lymphomas and thus help elucidate the genetics and pathophysiological mechanisms underlying the disease.

DNA-based sensor for real-time measurement of the enzymatic activity of human topoisomerase I.

Sensors capable of quantitative real-time measurements may present the easiest and most accurate way to study enzyme activities. Here we present a novel DNA-based sensor for specific and quantitative real-time measurement of the enzymatic activity of the essential human enzyme, topoisomerase I. The basic design of the sensor relies on two DNA strands that hybridize to form a hairpin structure with a fluorophore-quencher pair. The quencher moiety is released from the sensor upon reaction with human topoisomerase I thus enabling real-time optical measurement of enzymatic activity. The sensor is specific for topoisomerase I even in raw cell extracts and presents a simple mean of following enzyme kinetics using standard laboratory equipment such as a qPCR machine or fluorimeter. Human topoisomerase I is a well-known target for the clinically used anti-cancer drugs of the camptothecin family. The cytotoxic effect of camptothecins correlates directly with the intracellular topoisomerase I activity. We therefore envision that the presented sensor may find use for the prediction of cellular drug response. Moreover, inhibition of topoisomerase I by camptothecin is readily detectable using the presented DNA sensor, suggesting a potential application of the sensor for first line screening for potential topoisomerase I targeting anti-cancer drugs.

Prognostic value of KRAS mutations, TP53 mutations and PD-L1 expression among lung adenocarcinomas treated with immunotherapy.

AIMS: The aim of this study was to investigate the association between oncogenic alterations and programmed cell death ligand 1 (PD-L1) expression in lung adenocarcinomas, as well as the prognostic value of KRAS and/or TP53 mutations in patients treated with immunotherapy. METHODS: This study is a retrospective cohort study of 519 patients with lung adenocarcinomas analysed for mutations and PD-L1 expression. Data were collected from electronic pathology record system, next-generation sequencing system, and clinical databases. Association between mutations and PD-L1 expression was investigated, as well as survival statistics of the 65 patients treated with immunotherapy. RESULTS: 41% of the samples contained a KRAS mutation, predominantly together with mutations in TP53 (41%) or STK11 (10%). Higher expression of PD-L1 was seen among patients with KRAS mutations (p=0.002) and EGFR wild type (p=0.006). For patients treated with immunotherapy, there was no statistically significant difference for overall survival (OS) and progression-free survival (PFS) according to KRAS mutation status, TP53 mutation status or PD-L1 expression. The HR for concomitant mutations in TP53 and KRAS was 0.78 (95% CI 0.62 to 0.99) for OS and 0.43 (0.21 to 0.88) for PFS. Furthermore, concomitant TP53 and KRAS mutations predicted a better PFS (p=0.015) and OS (p=0.029) compared with no mutations or a single mutation in either TP53 or KRAS. CONCLUSION: Mutations in TP53 together with KRAS may serve as a potential biomarker for survival benefits with immunotherapy.

Cell-free chromatin immunoprecipitation can determine tumor gene expression in lung cancer patients.

Cell-free DNA (cfDNA) in blood plasma can be bound to nucleosomes that contain post-translational modifications representing the epigenetic profile of the cell of origin. This includes histone H3 lysine 36 trimethylation (H3K36me3), a marker of active transcription. We hypothesised that cell-free chromatin immunoprecipitation (cfChIP) of H3K36me3-modified nucleosomes present in blood plasma can delineate tumour gene expression levels. H3K36me3 cfChIP followed by targeted NGS (cfChIP-seq) was performed on blood plasma samples from non-small-cell lung cancer (NSCLC) patients (NSCLC, n = 8), small-cell lung cancer (SCLC) patients (SCLC, n = 4) and healthy controls (n = 4). H3K36me3 cfChIP-seq demonstrated increased enrichment of mutated alleles compared with normal alleles in plasma from patients with known somatic cancer mutations. Additionally, genes identified to be differentially expressed in SCLC and NSCLC tumours had concordant H3K36me3 cfChIP enrichment profiles in NSCLC (sensitivity = 0.80) and SCLC blood plasma (sensitivity = 0.86). Findings here expand the utility of cfDNA in liquid biopsies to characterise treatment resistance, cancer subtyping and disease progression.