Maurice P. H. M. Jansen

Background: Almost half of patients with colorectal liver metastases (CRLM) experience disease recurrence within one year after treatment with curative intent. To improve treatment, upfront identification of patients with CRLM at high risk of rapid disease recurrence is crucial. Methods: In this retrospective cohort-study, pretreatment ctDNA levels were determined by the modified fast aneuploidy screening test-sequencing system (mFast-SeqS) in 182 patients with resectable CRLM who did not receive perioperative chemotherapy. Resulting aneuploidy scores were dichotomized using a predefined threshold and associated with clinical outcome. Results: Of 182 analysed patients, 34 (19%) were classified as ctDNA high and 156 (81%) as ctDNA low. Recurrence-free (RFS) and overall survival (OS) were shorter in the ctDNA high versus low group, with 1-year RFS of 29% versus 52%, and 3-year OS of 48% versus 78% respectively (log-rank P = 0.029 and < 0.001 respectively). The cumulative incidence of multiorgan recurrence within the first year after local treatment was significantly higher in ctDNA-high patients (Gray’s test P < 0.001). Multivariable Cox regression analysis revealed that the aneuploidy score is independently associated with RFS (HR = 1.94; 95% c.i. 1.19 to 3.18) and multiorgan RFS up to 1 year (HR = 2.56; 95% c.i. 1.41 to 4.65) and OS up to 3 years (HR = 3.28; 95% c.i. 1.79 to 6.01). Conclusions: This study shows that mFast-SeqS provides an affordable and minimally invasive test for the upfront recognition of patients with CRLM at increased risk of rapid (multiorgan) disease recurrence.

HOTAIR and EZH2 have been identified to interact and to play a role in breast cancer development. In this retrospective clinical cohort study, HOTAIR lncRN

Around 30% of patients with hormone receptor-positive (HR+) breast cancer acquire resistance to endocrine therapy combined with cyclin-dependent kinase 4/6 inhibitors (CDK4/6i), which are first-line treatments in metastatic settings. Therefore, we aimed to identify loci associated with resistance to endocrine therapy and CDK4/6i; this was achieved using retroviral vectors, which randomly insert gene-disrupting elements into the genome, causing gene expression alterations and potentially leading to therapy resistance. ER-positive ZR75.1 breast cancer cells transduced with retroviral vectors were treated with endocrine (tamoxifen, fulvestrant) or CDK4/6i monotherapies (abemaciclib, palbociclib, ribociclib) or a combination of fulvestrant and ribociclib. DNA was extracted, and virus integration sites (VISs) were characterized according to the detection frequency and read depth using next-generation sequencing (VIS-NGS). Resistance-associated VIS loci were identified when differentially presented in treated samples compared to controls. Well-established tamoxifen resistance genes (BCAR1, BCAR3, EGFR) were detected, enabling the validation of our approach. Thirty-seven VIS loci were associated with resistance to fulvestrant and ribociclib monotherapies. Twenty of these loci were also identified as candidates for resistance to other CDK4/6i and to fulvestrant and ribociclib combination therapy, including TRPS1 and TRIM24—genes that are involved in resistance to endocrine therapy but have not yet been associated with resistance to CDK4/6i. The identification of unique and shared resistance-associated loci highlights the complexity of resistance pathways.

Retroviral transduction of cancer cell lines has been used to find genes related to therapy resistance. Characterization of virus integration sites (VIS) pinpointing these genes has been cumbersome. This study defines a sequencing-based protocol to rapidly characterize genomic loci near VIS. The protocol selectively amplifies VIS-genome junctions using the retroviral vector neomycin (NEO) gene, Genomic Walker Adapter approach, linker-mediated NEO-PCR (LM-NEO-PCR) or biotinylated NEO-capture, followed by long terminal repeats PCR (LTR-PCR). LTR-genome junctions were sequenced (NGS), reads mapped, quantified, and linked to genes. The protocol was tested on DNA from single clones holding twenty reported VIS loci and on multiplex and diluted clone DNA samples. Our VIS-NGS protocol enriched significantly (p < 0.02) more loci at high reads coverage in samples with VIS compared to negative controls. The protocol found seventeen reported VIS loci (85%) in single clone DNAs, of which fifteen loci (88%) were also detected in multiplex samples. Six loci were evaluated for all dilutions, with three loci detected at lowest 1% clone proportion. The protocol can be conducted in two weeks and successfully found almost all VIS loci in single VIS clones and detected half of the evaluated loci at low clone proportion.

Cell‐free circulating tumor DNA (ctDNA) has emerged as a promising biomarker for response evaluation in metastatic castration‐resistant prostate cancer (mCRPC). The current study evaluated the modified fast aneuploidy screening test‐sequencing system (mFast‐SeqS), a quick, tumor‐agnostic and affordable ctDNA assay that requires a small input of DNA, to generate a genome‐wide aneuploidy (GWA) score in mCRPC patients, and correlated this to matched metastatic tumor biopsies. In this prospective multicenter study, GWA scores were evaluated from blood samples of 196 mCRPC patients prior to treatment (baseline) with taxanes (docetaxel and cabazitaxel) and androgen receptor signaling inhibitors (ARSI; abiraterone and enzalutamide), and from 74 mCRPC patients at an early timepoint during treatment (early timepoint; median 21 days). Z ‐scores per chromosome arm were tested for their association with tumor tissue genomic alterations. We found that a high tumor load in blood (GWA ^high ) at baseline was associated with poor response to ARSI [HR: 2.63 (95% CI: 1.86–3.72) P < 0.001] but not to taxanes. Interestingly, GWA ^high score at the early timepoint was associated with poor response to both ARSIs [HR: 6.73 (95% CI: 2.60–17.42) P < 0.001] and taxanes [2.79 (95% CI: 1.34–5.78) P = 0.006]. A significant interaction in Cox proportional hazards analyses was seen when combining GWA status and type of treatment (at baseline P = 0.008; early timepoint P = 0.018). In summary, detection of ctDNA in blood by mFast‐SeqS is cheap, fast and feasible, and could be used at different timepoints as a potential predictor for outcome to ARSI and taxane treatment in mCRPC.

Activating mutations in the phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) gene have been detected often in solid tumors. Targeted therapy for mutant PIK3CA is now available in the clinic, making molecular diagnostics pivotal. Our aim was to design a multiplex digital PCR (dPCR) assay to evaluate the 4 most common PIK3CA hotspot mutations simultaneously to characterize and quantify these in liquid biopsies. A multiplex assay was developed to detect exon 9 p.E542K and p.E545K mutations, and exon 20 p.H1047L and p.H1047R mutations using the Stilla 3-color dPCR Naica system. The assay was evaluated on stock and pre-amplified DNA from cell lines with the above mutations as single and pooled samples, and on cell-free DNA (cfDNA) from healthy blood donors (HBDs) and breast cancer patients, to determine detection thresholds and diagnostic accuracy. The assay distinguished all 4 PIK3CA mutations in (cf)DNA, and also when dual mutations were present. Detection thresholds of stock and pre-amplified cfDNA samples were 0.11 and 0.40 copies/uL (cp/uL) for mutant copies concentration, and 0.003% and 0.68% for variant allele frequencies (VAFs), respectively. The assay confirmed the PIK3CA (mutation) status as defined by targeted next-generation sequencing (NGS) in 82 out of 96 patients that were mutant for PIK3CA, and in 11 out of 12 patients with wild-type PIK3CA. Our designed multiplex dPCR assay detected PIK3CA mutations with high accuracy in stock and pre-amplified cfDNA. Furthermore, it is affordable and demands less cfDNA input when compared to available uniplex dPCR assays and NGS analyses.

Background: Patients with advanced-stage epithelial ovarian cancer (EOC) receive treatment with a poly-ADP ribose-polymerase (PARP) inhibitor (PARPi) as maintenance therapy after surgery and chemotherapy. Unfortunately, many patients experience disease progression because of acquired therapy resistance. This study aims to characterize epigenetic and genomic changes in cell-free DNA (cfDNA) associated with PARPi resistance. Materials and Methods: Blood was taken from 31 EOC patients receiving PARPi therapy before treatment and at disease progression during/after treatment. Resistance was defined as disease progression within 6 months after starting PARPi and was seen in fifteen patients, while sixteen patients responded for 6 to 42 months. Blood cfDNA was evaluated via Modified Fast Aneuploidy Screening Test-Sequencing System (mFast-SeqS to detect aneuploidy, via Methylated DNA Sequencing (MeD-seq) to find differentially methylated regions (DMRs), and via shallow whole-genome and -exome sequencing (shWGS, exome-seq) to define tumor fractions and mutational signatures. Results: Aneuploid cfDNA was undetectable pre-treatment but observed in six patients post-treatment, in five resistant and one responding patient. Post-treatment ichorCNA analyses demonstrated in shWGS and exome-seq higher median tumor fractions in resistant (7% and 9%) than in sensitive patients (7% and 5%). SigMiner analyses detected predominantly mutational signatures linked to mismatch repair and chemotherapy. DeSeq2 analyses of MeD-seq data revealed three methylation signatures and more tumor-specific DMRs in resistant than in responding patients in both pre- and post-treatment samples (274 vs. 30 DMRs, 190 vs. 57 DMRs, Χ²-test p < 0.001). Conclusion: Our genome-wide Next-Generation Sequencing (NGS) analyses in PARPi-resistant patients identified epigenetic differences in blood before treatment, whereas genomic alterations were more frequently observed after progression. The epigenetic differences at baseline are especially interesting for further exploration as putative predictive biomarkers for PARPi resistance.

Molecular profiling may enable earlier detection of pancreatic cancer (PC) in high-risk individuals undergoing surveillance and allow for personalization of treatment. We hypothesized that the detection rate of DNA mutations is higher in pancreatic juice (PJ) than in plasma due to its closer contact with the pancreatic ductal system, from which pancreatic cancer cells originate, and higher overall cell-free DNA (cfDNA) concentrations. In this study, we included patients with pathology-proven PC or intraductal papillary mucinous neoplasm (IPMN) with high-grade dysplasia (HGD) from two prospective clinical trials (KRASPanc and PACYFIC) for whom both PJ and plasma were available. We performed next-generation sequencing on PJ, plasma, and tissue samples and described the presence (and concordance) of mutations in these biomaterials. This study included 26 patients (25 PC and 1 IPMN with HGD), of which 7 were women (27%), with a median age of 71 years (IQR 12) and a median BMI of 23 kg/m² (IQR 4). Ten patients with PC (40%) were (borderline) resectable at baseline. Tissue was available from six patients (resection n = 5, biopsy n = 1). A median volume of 2.9 mL plasma (IQR 1.0 mL) and 0.7 mL PJ (IQR 0.1 mL, p < 0.001) was used for DNA isolation. PJ had a higher median cfDNA concentration (2.6 ng/μL (IQR 4.2)) than plasma (0.29 ng/μL (IQR 0.40)). A total of 41 unique somatic mutations were detected: 24 mutations in plasma (2 KRAS, 15 TP53, 2 SMAD4, 3 CDKN2A 1 CTNNB1, and 1 PIK3CA), 19 in PJ (3 KRAS, 15 TP53, and 1 SMAD4), and 8 in tissue (2 KRAS, 2 CDKN2A, and 4 TP53). The mutation detection rate (and the concordance with tissue) did not differ between plasma and PJ. In conclusion, while the concentration of cfDNA was indeed higher in PJ than in plasma, the mutation detection rate was not different. A few cancer-associated genetic variants were detected in both biomaterials. Further research is needed to increase the detection rate and assess the performance and suitability of plasma and PJ for PC (early) detection.

Background: In breast cancer (BC), recurrent fusion genes of estrogen receptor alpha (ESR1) and AKAP12, ARMT1 and CCDC170 have been reported. In these gene fusions the ligand binding domain of ESR1 has been replaced by the transactivation domain of the fusion partner constitutively activating the receptor. As a result, these gene fusions can drive tumor growth hormone independently as been shown in preclinical models, but the clinical value of these fusions have not been reported. Here, we studied the prognostic and predictive value of different frequently reported ESR1 fusion transcripts in primary BC. Methods: We evaluated 732 patients with primary BC (131 ESR1-negative and 601 ESR1-positive cases), including two ER-positive BC patient cohorts: one cohort of 322 patients with advanced disease who received first-line endocrine therapy (ET) (predictive cohort), and a second cohort of 279 patients with lymph node negative disease (LNN) who received no adjuvant systemic treatment (prognostic cohort). Fusion gene transcript levels were measured by reverse transcriptase quantitative PCR. The presence of the different fusion transcripts was associated, in uni- and multivariable Cox regression analysis taking along current clinico-pathological characteristics, to progression free survival (PFS) during first-line endocrine therapy in the predictive cohort, and disease- free survival (DFS) and overall survival (OS) in the prognostic cohort. Results: The ESR1-CCDC170 fusion transcript was present in 27.6% of the ESR1-positive BC subjects and in 2.3% of the ESR1-negative cases. In the predictive cohort, none of the fusion transcripts were associated with response to first-line ET. In the prognostic cohort, the median DFS and OS were respectively 37 and 93 months for patients with an ESR1-CCDC170 exon 8 gene fusion transcript and respectively 91 and 212 months for patients without this fusion transcript. In a multivariable analysis, this ESR1-CCDC170 fusion transcript was an independent prognostic factor for DFS (HR) (95% confidence interval (CI): 1.8 (1.2–2.8), P = 0.005) and OS (HR (95% CI: 1.7 (1.1–2.7), P = 0.023). Conclusions: Our study shows that in primary BC only ESR1-CCDC170 exon 8 gene fusion transcript carries prognostic value. None of the ESR1 fusion transcripts, which are considered to have constitutive ER activity, was predictive for outcome in BC with advanced disease treated with endocrine treatment.

Background: In breast cancer (BC), recurrent fusion genes of estrogen receptor alpha (ESR1) and AKAP12, ARMT1 and CCDC170 have been reported. In these gene fusions the ligand binding domain of ESR1 has been replaced by the transactivation domain of the fusion partner constitutively activating the receptor. As a result, these gene fusions can drive tumor growth hormone independently as been shown in preclinical models, but the clinical value of these fusions have not been reported. Here, we studied the prognostic and predictive value of different frequently reported ESR1 fusion transcripts in primary BC. Methods: We evaluated 732 patients with primary BC (131 ESR1-negative and 601 ESR1-positive cases), including two ER-positive BC patient cohorts: one cohort of 322 patients with advanced disease who received first-line endocrine therapy (ET) (predictive cohort), and a second cohort of 279 patients with lymph node negative disease (LNN) who received no adjuvant systemic treatment (prognostic cohort). Fusion gene transcript levels were measured by reverse transcriptase quantitative PCR. The presence of the different fusion transcripts was associated, in uni- and multivariable Cox regression analysis taking along current clinic-pathological characteristics, to progression free survival (PFS) during first-line endocrine therapy in the predictive cohort, and disease- free survival (DFS) and overall survival (OS) in the prognostic cohort.Results: The ESR1-CCDC170 fusion transcript was present in 27.6% of the ESR1-positive BC subjects and in 2.3% of the ESR1-negative cases. In the predictive cohort, none of the fusion transcripts were associated with response to first-line ET. In the prognostic cohort, the median DFS and OS were respectively 37 and 93 months for patients with an ESR1-CCDC170 exon 8 gene fusion transcript and respectively 91 and 212 months for patients without this fusion transcript. In a multivariable analysis, this ESR1-CCDC170 fusion transcript was an independent prognostic factor for DFS (HR) (95% confidence interval (CI): 1.8 (1.2–2.8), P=0.005) and OS (HR (95% CI: 1.7 (1.1–2.7), P=0.023). Conclusions: Our study shows that in primary BC only ESR1-CCDC170 exon 8 gene fusion transcript carries prognostic value. None of the ESR1 fusion transcripts, which are considered to have constitutive ER activity, was predictive for outcome in BC with advanced disease treated with endocrine treatment.