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Advances in therapeutic approaches for melanoma urge the need for biomarkers that can identify patients at risk for recurrence and to guide treatment. The potential use of liquid biopsies in identifying biomarkers is increasingly being recognized. Here, we present a head‐to‐head comparison of several techniques to analyze circulating tumor cells (CTCs) and cell‐free DNA (cfDNA) in 20 patients with metastatic melanoma. In this study, we investigated whether diagnostic leukapheresis (DLA) combined with multimarker flow cytometry (FCM) increased the detection of CTCs in blood compared to the CellSearch platform. Additionally, we characterized cfDNA at the level of somatic mutations, extent of aneuploidy and genome‐wide DNA methylation. Both CTCs and cfDNA measures were compared to tumor markers and extracranial tumor burden on radiological imaging. Compared to the CellSearch method applied on peripheral blood, DLA combined with FCM increased the proportion of patients with detectable CTCs from 35% to 70% (P = 0.06). However, the median percentage of cells that could be recovered by the DLA procedure was 29%. Alternatively, cfDNA mutation and methylation analysis detected tumor load in the majority of patients (90% and 93% of samples successfully analyzed, respectively). The aneuploidy score was positive in 35% of all patients. From all tumor measurements in blood, lactate dehydrogenase (LDH) levels were significantly correlated to variant allele frequency (P = 0.004). Furthermore, the presence of CTCs in DLA was associated with tumor burden (P

Abstract Spatial distribution of intra-tumoral immune cell populations is considered a critical determinant of tumor evolution and response to therapy. The accurate and systemic search for contexture-based predictors would be accelerated by methods that allow interactive visualization and interrogation of tumor micro-environments (TME), independent of image acquisition platforms. To this end, we have developed the TME-Analyzer, a new image analysis tool, which we have benchmarked against 2 software tools regarding densities and networks of immune effector cells using multiplexed immune-fluorescent images of triple negative breast cancer (TNBC). With the TME-Analyzer we have identified a 10-parameter classifier, predominantly featuring cellular distances, that significantly predicted overall survival, and which was validated using multiplexed ion beam time of flight images from an independent cohort. In conclusion, the TME-Analyzer enabled accurate interactive analysis of the spatial immune phenotype from different imaging platforms as well as enhanced utility and aided the discovery of contextual predictors towards the survival of TNBC patients.

Abstract Background In breast cancer, the advent of anti-HER2 therapies has made HER2+ tumors a highly relevant subgroup. However, the exact characteristics which prohibit clinical response to anti-HER2 therapies and drive disease progression are not yet fully known. Integrative whole-genome and transcriptomic sequencing data from both primary and metastatic HER2-positive breast cancer will enhance our understanding of underlying biological processes. Methods Here, we used WGS and RNA sequencing data of 700 metastatic breast tumors, of which 68 being HER2+, to search for specific genomic features of HER2+ disease and therapy resistance. Furthermore, we integrated results with transcriptomic data to associate tumors exhibiting a HER2+-specific gene expression profile with ERBB2 mutation status, prior therapy and relevant gene expression signatures. Results Overall genomic profiles of primary and metastatic HER2+ breast cancers were similar, and no specific acquired genomics traits connected to prior anti-HER2 treatment were observed. However, specific genomic features were predictive of progression-free survival on post-biopsy anti-HER2 treatment. Furthermore, a HER2-driven expression profile grouped HER2-amplified tumors with ERBB2-mutated cases and cases without HER2 alterations. The latter were reported as ER positive in primary disease, but the metastatic biopsy showed low ESR1 expression and upregulation of the MAPK pathway, suggesting transformation to ER independence. Conclusions In summary, although the quantity of variants increased throughout HER2-positive breast cancer progression, the genomic composition remained largely consistent, thus yielding no new major processes beside those already operational in primary disease. Our results suggest that integrated genomic and transcriptomic analyses may be key in establishing therapeutic options.

Multiple prognostic biomarkers, including circulating tumour cell (CTC) counts, exist in metastatic castration‐resistant prostate cancer (mCRPC) patients, but none of them have been implemented into daily clinical care. The modified fast aneuploidy screening test‐sequencing system (mFast‐SeqS), which yields a genome‐wide aneuploidy score, is able to reflect the fraction of cell‐free tumour DNA (ctDNA) within cell‐free DNA (cfDNA) and may be a promising biomarker in mCRPC. In this study, we investigated the prognostic value of dichotomized aneuploidy scores (

Abstract Obesity is associated with an increased risk of developing breast cancer (BC) and worse prognosis in BC patients, yet its impact on BC biology remains understudied in humans. This study investigates how the biology of untreated primary BC differs according to patients’ body mass index (BMI) using data from >2,000 patients. We identify several genomic alterations that are differentially prevalent in overweight or obese patients compared to lean patients. We report evidence supporting an ageing accelerating effect of obesity at the genetic level. We show that BMI-associated differences in bulk transcriptomic profile are subtle, while single cell profiling allows detection of more pronounced changes in different cell compartments. These analyses further reveal an elevated and unresolved inflammation of the BC tumor microenvironment associated with obesity, with distinct characteristics contingent on the estrogen receptor status. Collectively, our analyses imply that obesity is associated with an inflammaging-like phenotype. We conclude that patient adiposity may play a significant role in the heterogeneity of BC and should be considered for BC treatment tailoring.

Abstract This prospective cohort study reports aneuploidy score by mFast-SeqS as a strong prognostic marker in MBC patients. mFAST-SeqS is an affordable and easily implementable method for the assessment of total ctDNA levels and, as such, provides an alternative prognostic tool. One mixed cohort (cohort A, n = 45) starting any type of treatment in any line of therapy and one larger cohort (cohort B, n = 129) consisting of patients starting aromatase inhibitors (AI) as first-line therapy were used. mFAST-SeqS was performed using plasma of blood in which CTCs (CellSearch) were enumerated. The resulting aneuploidy score was correlated with categorized CTC count and associated with outcome. The aneuploidy score was significantly correlated with CTC count, but discordance was observed in 31.6% when applying cut-offs of 5. In both cohorts, aneuploidy score was a significant prognostic marker for both PFS and OS. In the Cox regression models, the HR for aneuploidy score for PFS was 2.52 (95% CI: 1.56–4.07), and the HR for OS was 2.37 (95% CI: 1.36–4.14). Results presented here warrant further investigations into the clinical utility of this marker in MBC patients.

Abstract Next generation sequencing of cell-free DNA (cfDNA) is a promising method for treatment monitoring and therapy selection in metastatic breast cancer (MBC). However, distinguishing tumor-specific variants from sequencing artefacts and germline variation with low false discovery rate is challenging when using large targeted sequencing panels covering many tumor suppressor genes. To address this, we built a machine learning model to remove false positive variant calls and augmented it with additional filters to ensure selection of tumor-derived variants. We used cfDNA of 70 MBC patients profiled with both the small targeted Oncomine breast panel (Thermofisher) and the much larger Qiaseq Human Breast Cancer Panel (Qiagen). The model was trained on the panels’ common regions using Oncomine hotspot mutations as ground truth. Applied to Qiaseq data, it achieved 35% sensitivity and 36% precision, outperforming basic filtering. For 20 patients we used germline DNA to filter for somatic variants and obtained 245 variants in total, while our model found seven variants, of which six were also detected using the germline strategy. In ten tumor-free individuals, our method detected in total one (potentially germline) variant, in contrast to 521 variants detected without our model. These results indicate that our model largely detects somatic variants.

Abstract Background CHEK2 c.1100delC was the first moderate-risk breast cancer (BC) susceptibility allele discovered. Despite several genomic, transcriptomic and functional studies, however, it is still unclear how exactly CHEK2 c.1100delC promotes tumorigenesis. Since the mutational landscape of a tumor reflects the processes that have operated on its development, the aim of this study was to uncover the somatic genomic landscape of CHEK2-associated BC. Methods We sequenced primary BC (pBC) and normal genomes of 20 CHEK2 c.1100delC mutation carriers as well as their pBC transcriptomes. Including pre-existing cohorts, we exhaustively compared CHEK2 pBC genomes to those from BRCA1/2 mutation carriers, those that displayed homologous recombination deficiency (HRD) and ER− and ER+ pBCs, totaling to 574 pBC genomes. Findings were validated in 517 metastatic BC genomes subdivided into the same subgroups. Transcriptome data from 168 ER+ pBCs were used to derive a TP53-mutant gene expression signature and perform cluster analysis with CHEK2 BC transcriptomes. Finally, clinical outcome of CHEK2 c.1100delC carriers was compared with BC patients displaying somatic TP53 mutations in two well-described retrospective cohorts totaling to 942 independent pBC cases. Results BC genomes from CHEK2 mutation carriers were most similar to ER+ BC genomes and least similar to those of BRCA1/2 mutation carriers in terms of tumor mutational burden as well as mutational signatures. Moreover, CHEK2 BC genomes did not show any evidence of HRD. Somatic TP53 mutation frequency and the size distribution of structural variants (SVs), however, were different compared to ER+ BC. Interestingly, BC genomes with bi-allelic CHEK2 inactivation lacked somatic TP53 mutations and transcriptomic analysis indicated a shared biology with TP53 mutant BC. Moreover, CHEK2 BC genomes had an increased frequency of > 1 Mb deletions, inversions and tandem duplications with peaks at specific sizes. The high chromothripsis frequency among CHEK2 BC genomes appeared, however, not associated with this unique SV size distribution profile. Conclusions CHEK2 BC genomes are most similar to ER+ BC genomes, but display unique features that may further unravel CHEK2-driven tumorigenesis. Increased insight into this mechanism could explain the shorter survival of CHEK2 mutation carriers that is likely driven by intrinsic tumor aggressiveness rather than endocrine resistance.

Abstract Grainyhead like 2 (GRHL2) is an essential transcription factor for development and function of epithelial tissues. It has dual roles in cancer by supporting tumor growth while suppressing epithelial to mesenchymal transitions (EMT). GRHL2 cooperates with androgen and estrogen receptors (ER) to regulate gene expression. We explore genome wide GRHL2 binding sites conserved in three ER⍺/GRHL2 positive luminal breast cancer cell lines by ChIP-Seq. Interaction with the ER⍺/FOXA1/GATA3 complex is observed, however, only for a minor fraction of conserved GRHL2 peaks. We determine genome wide transcriptional dynamics in response to loss of GRHL2 by nascent RNA Bru-seq using an MCF7 conditional knockout model. Integration of ChIP- and Bru-seq pinpoints candidate direct GRHL2 target genes in luminal breast cancer. Multiple connections between GRHL2 and proliferation are uncovered, including transcriptional activation of ETS and E2F transcription factors. Among EMT-related genes, direct regulation of CLDN4 is corroborated but several targets identified in other cells (including CDH1 and ZEB1) are ruled out by both ChIP- and Bru-seq as being directly controlled by GRHL2 in luminal breast cancer cells. Gene clusters correlating positively (including known GRHL2 targets such as ErbB3, CLDN4/7) or negatively (including TGFB1 and TGFBR2) with GRHL2 in the MCF7 knockout model, display similar correlation with GRHL2 in ER positive as well as ER negative breast cancer patients. Altogether, this study uncovers gene sets regulated directly or indirectly by GRHL2 in luminal breast cancer, identifies novel GRHL2-regulated genes, and points to distinct GRHL2 regulation of EMT in luminal breast cancer cells. Video Abstract

The risk of germline copy number variants (CNVs) in BRCA1 and BRCA2 pathogenic variant carriers in breast cancer is assessed, with CNVs overlapping SULT1A1 decreasing breast cancer risk in BRCA1 carriers.

Colorectal cancer can lead to the development of peritoneal metastases, which are associated with worse disease outcome. Here, the authors characterize peritoneal metastases from 52 patients using RNA-seq and mutational sequencing and show a distinct molecular subtype.

Intrapatient tumour heterogeneity is likely a major determinant of clinical outcome in cancer patients. To assess heterogeneity in a minimally invasive manner, methods to perform single circulating tumour cell (CTC) genomics at high resolution are necessary. However, due to the rarity of CTCs, development of such methods is challenging. Here, we developed a modular single CTC analysis pipeline to assess intrapatient heterogeneity by copy number (CN) profiling. To optimize this pipeline, spike‐in experiments using MCF‐7 breast cancer cells were performed. The VyCAP puncher system was used to isolate single cells. The quality of whole genome amplification (WGA) products generated by REPLI‐g and Ampli1™ methods, as well as the results from the Illumina Truseq and the Ampli1™ LowPass library preparation techniques, was compared. Moreover, a bioinformatic pipeline was designed to generate CN profiles from single CTCs. The optimal combination of Ampli1™ WGA and Illumina Truseq library preparation was successfully validated on patient‐derived CTCs. In conclusion, we developed a novel modular pipeline to isolate single CTCs and subsequently generate detailed patient‐derived CN profiles that allow assessment of intrapatient heterogeneity in future studies.

Abstract Currently, the human epidermal growth factor receptor 2 (HER2) status of breast cancer is classified dichotomously as negative or positive to select patients for HER2-targeted therapy. However, with the introduction of novel treatment options, it is important to get more insight in the biology of cancers with low HER2 expression. Therefore, we studied several clinicopathologic characteristics in relation to the level of HER2 expression (HER2- versus HER2low). We used a well-documented cohort of breast cancer patients (n = 529), with available tissue microarrays and Affymetrix mRNA expression data. HER2 status was scored as negative (immunohistochemistry 0) or low (immunohistochemistry 1 + or 2 + without amplification). We associated HER2 status with several clinicopathologic characteristics, gene-expression data and survival, stratified for estrogen receptor (ER) status. Overall, breast cancers were scored as HER2- (n = 429) or HER2low (n = 100). Within the ER+ cohort (n = 305), no significant associations were found between the HER2 groups and clinicopathologic features. However, HER2low tumors showed several differentially expressed genes compared to HER2- cases, including genes that are associated with worse outcome and depletion of immunity. In ER- cases (n = 224), HER2low status was significantly associated with increased regional nodal positivity, lower density of tumor infiltrating lymphocyte and a lower protein expression of Ki-67 and EGFR compared to HER2- cases. After multivariate analysis, only density of tumor infiltrating lymphocytes remained significantly associated with HER2low status (P = 0.035). No difference in survival was observed between HER2low and HER2- patients, neither in the ER+ nor ER- cohort. In conclusion, our data suggests that HER2low breast cancer is associated with a lower immune response compared to HER2- breast cancer.

Second‐line treatment with immune checkpoint inhibition in patients with metastatic urothelial cancer (mUC) has a low success rate (~ 20%). Circulating tumour‐derived DNA (ctDNA) levels may guide patient stratification, provided that an affordable and robust assay is available. Here, we investigate whether the modified fast aneuploidy screening test‐sequencing system (mFast‐SeqS) may provide such an assay. To this end, mFast‐SeqS was performed on cell‐free DNA (cfDNA) from 74 patients with mUC prior to treatment with pembrolizumab. Results were associated with corresponding tissue‐based profiles, plasma‐based variant allele frequencies (VAFs) and clinical response. We found that plasma‐derived mFast‐SeqS‐based aneuploidy scores significantly correlated with those observed in the corresponding tumour tissue as well as with the ctDNA level in the plasma. In multivariate logistic regression analysis, a high aneuploidy score was independently associated with lack of clinical benefit from treatment with pembrolizumab. In conclusion, mFast‐SeqS provides a patient‐friendly, high‐throughput and affordable method to estimate ctDNA level. Following independent validation, this test could be used to stratify mUC patients for response prior to the initiation of treatment with pembrolizumab.

Circulating tumour DNA (ctDNA) is a potential biomarker that could contribute to more judicious patient selection for personalised treatment. This review and meta-analysis gives an overview of the current knowledge in the literature investigating the value of ctDNA in patients with colorectal liver metastases (CRLM). A systematic search was conducted in electronic databases for studies published prior to the 26th of May 2023. Studies investigating the association between ctDNA and oncological outcomes in patients undergoing curative-intent local therapy for CRLM were included. Meta-analyses were performed to pool hazard ratios (HR) for the recurrence-free survival (RFS) and overall survival (OS). A total of eleven studies were included and nine were eligible for meta-analyses. Patients with detectable ctDNA after surgery experienced a significantly higher chance of recurrence (HR 3.12, 95% CI 2.27–4.28, p < 0.000010) and shorter OS (HR 5.04, 95% CI 2.53–10.04, p < 0.00001) compared to patients without detectable ctDNA. A similar association for recurrence was found in patients with detectable ctDNA after the completion of adjuvant therapy (HR 6.39, 95% CI 2.13–19.17, p < 0.0009). The meta-analyses revealed no association between detectable ctDNA before surgery and the RFS and OS. These meta-analyses demonstrate the strong association between detectable ctDNA after treatment and oncological outcomes in CRLM patients.

Abstract 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.

BackgroundCirculating tumour DNA (ctDNA) has been established as a promising (prognostic) biomarker with the potential to personalise treatment in cancer patients. The objective of this systematic review is to provide an overview of the current literature and the future perspectives of ctDNA in non-metastatic rectal cancer.MethodsA comprehensive search for studies published prior to the 4th of October 2022 was conducted in Embase, Medline, Cochrane, Google scholar, and Web of Science. Only peer-reviewed original articles and ongoing clinical trials investigating the association between ctDNA and oncological outcomes in non-metastatic rectal cancer patients were included. Meta-analyses were performed to pool hazard ratios (HR) for recurrence-free survival (RFS).ResultsA total of 291 unique records were screened, of which 261 were original publications and 30 ongoing trials. Nineteen original publications were reviewed and discussed, of which seven provided sufficient data for meta-analyses on the association between the presence of post-treatment ctDNA and RFS. Results of the meta-analyses demonstrated that ctDNA analysis can be used to stratify patients into very high and low risk groups for recurrence, especially when detected after neoadjuvant treatment (HR for RFS: 9.3 [4.6 – 18.8]) and after surgery (HR for RFS: 15.5 [8.2 – 29.3]). Studies investigated different types of assays and used various techniques for the detection and quantification of ctDNA.ConclusionsThis literature overview and meta-analyses provide evidence for the strong association between ctDNA and recurrent disease. Future research should focus on the feasibility of ctDNA-guided treatment and follow-up strategies in rectal cancer. A blueprint for agreed-upon timing, preprocessing, and assay techniques is needed to empower adaptation of ctDNA into daily practice.

Consensus molecular subtypes (CMSs) can guide precision treatment of colorectal cancer (CRC). We aim to identify methylation markers to distinguish between CMS2 and CMS3 in patients with CRC, for which an easy test is currently lacking. To this aim, fresh‐frozen tumor tissue of 239 patients with stage I‐III CRC was analyzed. Methylation profiles were obtained using the Infinium HumanMethylation450 BeadChip. We performed adaptive group‐regularized logistic ridge regression with post hoc group‐weighted elastic net marker selection to build prediction models for classification of CMS2 and CMS3. The Cancer Genome Atlas (TCGA) data were used for validation. Group regularization of the probes was done based on their location either relative to a CpG island or relative to a gene present in the CMS classifier, resulting in two different prediction models and subsequently different marker panels. For both panels, even when using only five markers, accuracies were > 90% in our cohort and in the TCGA validation set. Our methylation marker panel accurately distinguishes between CMS2 and CMS3. This enables development of a targeted assay to provide a robust and clinically relevant classification tool for CRC patients.

SPEN is a key player in initiation of X chromosome inactivation. Here, the authors show that SPEN is required for Xist-mediated silencing of its own antisense regulator Tsix.

Only a subset of triple negative breast cancer patients respond to immunotherapy. Here, the authors analysed spatial immune contextures, which can be captured by a gene classifier, in relation to genomic alterations, mechanisms of T cell evasion and response to anti-PD1 treatment.

Abstract Background Given the high heterogeneity among breast tumors, associations between common germline genetic variants and survival that may exist within specific subgroups could go undetected in an unstratified set of breast cancer patients. Methods We performed genome-wide association analyses within 15 subgroups of breast cancer patients based on prognostic factors, including hormone receptors, tumor grade, age, and type of systemic treatment. Analyses were based on 91,686 female patients of European ancestry from the Breast Cancer Association Consortium, including 7531 breast cancer-specific deaths over a median follow-up of 8.1 years. Cox regression was used to assess associations of common germline variants with 15-year and 5-year breast cancer-specific survival. We assessed the probability of these associations being true positives via the Bayesian false discovery probability (BFDP

Abstract Here, we describe a novel approach for rapid discovery of a set of tumor-specific genomic structural variants (SVs), based on a combination of low coverage cancer genome sequencing using Oxford Nanopore with an SV calling and filtering pipeline. We applied the method to tumor samples of high-grade ovarian and prostate cancer patients and validated on average ten somatic SVs per patient with breakpoint-spanning PCR mini-amplicons. These SVs could be quantified in ctDNA samples of patients with metastatic prostate cancer using a digital PCR assay. The results suggest that SV dynamics correlate with and may improve existing treatment-response biomarkers such as PSA. https://github.com/UMCUGenetics/SHARC .

APOBEC3B (A3B) is aberrantly overexpressed in a subset of breast cancers, where it associates with advanced disease, poor prognosis, and treatment resistance, yet the causes of A3B dysregulation in breast cancer remain unclear. Here, A3B mRNA and protein expression levels were quantified in different cell lines and breast tumors and related to cell cycle markers using RT-qPCR and multiplex immunofluorescence imaging. The inducibility of A3B expression during the cell cycle was additionally addressed after cell cycle synchronization with multiple methods. First, we found that A3B protein levels within cell lines and tumors are heterogeneous and associate strongly with the proliferation marker Cyclin B1 characteristic of the G2/M phase of the cell cycle. Second, in multiple breast cancer cell lines with high A3B, expression levels were observed to oscillate throughout the cell cycle and again associate with Cyclin B1. Third, induction of A3B expression is potently repressed throughout G0/early G1, likely by RB/E2F pathway effector proteins. Fourth, in cells with low A3B, induction of A3B through the PKC/ncNF-κB pathway occurs predominantly in actively proliferating cells and is largely absent in cells arrested in G0. Altogether, these results support a model in which dysregulated A3B overexpression in breast cancer is the cumulative result of proliferation-associated relief from repression with concomitant pathway activation during the G2/M phase of the cell cycle.

Abstract Background Triple negative breast cancer (TNBC) is an aggressive subtype of breast cancer with limited therapeutic opportunities. Recently, splicing factors have gained attention as potential targets for cancer treatment. Here we systematically evaluated the role of RNA splicing factors in TNBC cell proliferation. Methods In this study, we performed an RNAi screen targeting 244 individual splicing factors to systematically evaluate their role in TNBC cell proliferation. For top candidates, mechanistic insight was gained using amongst others western blot, PCR, FACS, molecular imaging and cloning. Pulldown followed by mass spectrometry were used to determine protein-protein interactions and patient-derived RNA sequencing data was used relate splicing factor expression levels to proliferation markers. Results We identified nine splicing factors, including SNRPD2, SNRPD3 and NHP2L1, of which depletion inhibited proliferation in two TNBC cell lines by deregulation of sister chromatid cohesion (SCC) via increased sororin intron 1 retention and down-regulation of SMC1, MAU2 and ESPL1. Protein-protein interaction analysis of SNRPD2, SNRPD3 and NHP2L1 identified that seven out of the nine identified splicing factors belong to the same spliceosome complex including novel component SUN2 that was also critical for efficient sororin splicing. Finally, sororin transcript levels are highly correlated to various proliferation markers in BC patients. Conclusion We systematically determined splicing factors that control proliferation of breast cancer cells through a mechanism that involves effective sororin splicing and thereby appropriate sister chromatid cohesion. Moreover, we identified SUN2 as an important new spliceosome complex interacting protein that is critical in this process. We anticipate that deregulating sororin levels through targeting of the relevant splicing factors might be a potential strategy to treat TNBC.

Abstract Background Extracellular vesicles (EVs) are actively secreted by cells into body fluids and contain nucleic acids of the cells they originate from. The goal of this study was to detect circulating tumor-derived EVs (ctEVs) by mutant mRNA transcripts (EV-RNA) in plasma of patients with solid cancers and compare the occurrence of ctEVs with circulating tumor DNA (ctDNA) in cell-free DNA (cfDNA). Methods For this purpose, blood from 20 patients and 15 healthy blood donors (HBDs) was collected in different preservation tubes (EDTA, BCT, CellSave) and processed into plasma within 24 h from venipuncture. EVs were isolated with the ExoEasy protocol from this plasma and from conditioned medium of 6 cancer cell lines and characterized according to MISEV2018-guidelines. RNA from EVs was isolated with the ExoRNeasy protocol and evaluated for transcript expression levels of 96 genes by RT-qPCR and genotyped by digital PCR. Results Our workflow applied on cell lines revealed a high concordance between cellular mRNA and EV-RNA in expression levels as well as variant allele frequencies for PIK3CA, KRAS and BRAF. Plasma CD9-positive EV and GAPDH EV-RNA levels were significantly different between the preservation tubes. The workflow detected only ctEVs with mutant transcripts in plasma of patients with high amounts (> 20%) of circulating tumor DNA (ctDNA). Expression profiling showed that the EVs from patients resemble healthy donors more than tumor cell lines supporting that most EVs are derived from healthy tissue. Conclusions We provide a workflow for ctEV detection by spin column-based generic isolation of EVs and PCR-based measurement of gene expression and mutant transcripts in EV-RNA derived from cancer patients’ blood plasma. This workflow, however, detected tumor-specific mutations in blood less often in EV-RNA than in cfDNA.

Abstract Screening for effective candidate drugs for breast cancer has shifted from two-dimensional (2D) to three-dimensional (3D) cultures. Here we systematically compared the transcriptomes of these different culture conditions by RNAseq of 14 BC cell lines cultured in both 2D and 3D conditions. All 3D BC cell cultures demonstrated increased mitochondrial metabolism and downregulated cell cycle programs. Luminal BC cells in 3D demonstrated overall limited reprogramming. 3D basal B BC cells showed increased expression of extracellular matrix (ECM) interaction genes, which coincides with an invasive phenotype not observed in other BC cells. Genes downregulated in 3D were associated with metastatic disease progression in BC patients, including cyclin dependent kinases and aurora kinases. Furthermore, the overall correlation of the cell line transcriptome to the BC patient transcriptome was increased in 3D cultures for all TNBC cell lines. To define the most optimal culture conditions to study the oncogenic pathway of interest, an open source bioinformatics strategy was established.

Breast cancer risk for BRCA1/BRCA2 mutation carriers varies depending on other genetic factors. Here, the authors perform a case-only genome-wide association study and highlight novel loci associated with breast cancer risk for BRCA1/BRCA2 mutation carriers.

Quantification of tumor‐specific variants (TSVs) in cell‐free DNA is rapidly evolving as a prognostic and predictive tool in patients with cancer. Currently, both variant allele frequency (VAF) and number of mutant molecules per mL plasma are used as units of measurement to report those TSVs. However, it is unknown to what extent both units of measurement agree and what are the factors underlying an existing disagreement. To study the agreement between VAF and mutant molecules in current clinical studies, we analyzed 1116 TSVs from 338 patients identified with next‐generation sequencing (NGS) or digital droplet PCR (ddPCR). On different study cohorts, a Deming regression analysis was performed and its 95% prediction interval was used as surrogate for the limits of agreement between VAF and number of mutant molecules per mL and to identify outliers. VAF and number of mutant molecules per mL plasma yielded greater agreement when using ddPCR than NGS. In case of discordance between VAF and number of mutant molecules per mL, insufficient molecular coverage in NGS and high cell‐free DNA concentration were the main responsible factors. We propose several optimization steps needed to bring monitoring of TSVs in cell‐free DNA to its full potential.

Circulating tumor cells (CTCs) in the blood of cancer patients are of high clinical relevance. Since detection and isolation of CTCs often rely on cell dimensions, knowledge of their size is key. We analyzed the median CTC size in a large cohort of breast (BC), prostate (PC), colorectal (CRC), and bladder (BLC) cancer patients. Images of patient‐derived CTCs acquired on cartridges of the FDA‐cleared CellSearch® method were retrospectively collected and automatically re‐analyzed using the accept software package. The median CTC diameter (μm) was computed per tumor type. The size differences between the different tumor types and references (tumor cell lines and leukocytes) were nonparametrically tested. A total of 1962 CellSearch® cartridges containing 71 612 CTCs were included. In BC, the median computed diameter (CD) of patient‐derived CTCs was 12.4 μm vs 18.4 μm for cultured cell line cells. For PC, CDs were 10.3 μm for CTCs vs 20.7 μm for cultured cell line cells. CDs for CTCs of CRC and BLC were 7.5 μm and 8.6 μm, respectively. Finally, leukocytes were 9.4 μm. CTC size differed statistically significantly between the four tumor types and between CTCs and the reference data. CTC size differences between tumor types are striking and CTCs are smaller than cell line tumor cells, whose size is often used as reference when developing CTC analysis methods. Based on our data, we suggest that the size of CTCs matters and should be kept in mind when designing and optimizing size‐based isolation methods.

Molecular landscapes of metastatic colorectal cancers (mCRC) have often been restricted to coding regions or low numbers of patients. Here the authors present a whole-genome landscape of 429 mCRC patients, revealing the mutational impact of prior therapies and potential actionable targets.

Prostate cancer is the most dominant male malignancy worldwide. The clinical presentation of prostate cancer ranges from localized indolent to rapidly progressing lethal metastatic disease. Despite a decline in death rate over the past years, with the advent of early diagnosis and new treatment options, challenges remain towards the management of metastatic prostate cancer, particularly metastatic castration sensitive prostate cancer (mCSPC) and castration resistant prostate cancer (mCRPC). Current treatments involve a combination of chemotherapy with androgen deprivation therapy and/or androgen receptor signalling inhibitors. However, treatment outcomes are heterogeneous due to significant tumor heterogeneity indicating a need for better prognostic biomarkers to identify patients with poor outcomes. Liquid biopsy has opened a plethora of opportunities from early diagnosis to (personalized) therapeutic disease interventions. In this review, we first provide recent insights about (metastatic) prostate cancer and its current treatment landscape. We highlight recent studies involving various circulating biomarkers such as circulating tumor cells, genetic markers, circulating nucleic acids, extracellular vesicles, tumor-educated platelets, and the secretome from (circulating) tumor cells and tumor microenvironment in metastatic prostate cancer. The comprehensive array of biomarkers can provide a powerful approach to understanding the spectrum of prostate cancer disease and guide in developing improved and personalized treatments for patients.

Abstract New therapies are an urgent medical need in all breast cancer subgroups. Metabotropic glutamate receptor 1 (mGluR1) is suggested as a potential new molecular target. We examined the prevalence mGluR1 expression in different clinically relevant breast cancer subgroups and determined its association with prognosis. In this retrospective cohort, 394 consecutive primary breast cancer tissues were incorporated into a tissue microarray and immunohistochemically stained for mGluR1. The prevalence of mGluR1 protein expression in different breast cancer subgroups was evaluated and correlated with metastasis-free survival (MFS) and overall survival (OS). In total, 56% (n = 219) breast cancer tissues had mGluR1 expression. In estrogen receptor (ER)-negative tumors, 31% (n = 18/58) had mGluR1 expression that was significantly associated with MFS (HR 5.00, 95% CI 1.03–24.35, p = 0.046) in multivariate analysis, independently from other prognostic factors. Of the 44 triple-negative breast cancer (TNBC), 25% (n = 11) expressed mGluR1. mGluR1 expression in TNBC was significantly associated with shorter MFS (HR 8.60, 95% CI 1.06–20.39, p = 0.044) and with poor OS (HR 16.07, 95% CI 1.16–223.10, p = 0.039). In conclusion, mGluR1 is frequently expressed in breast cancer. In ER-negative breast cancer and in TNBC mGluR1 protein expression is an unfavorable prognostic marker. This study provides rationale to explore mGluR1 as a novel target for breast cancer treatment, especially for the more aggressive TNBC.

Cancers deficient in homologous recombination can benefit from treatment with poly ADP-ribose polymerase (PARP) inhibitors. Here, the authors generated a classifier that can predict homologous recombination deficiency from genomic data and suggest several cancer types that may benefit from PARP inhibitor treatment.

Abstract Background Breast cancer (BC) immune infiltrates play a critical role in tumor progression and response to treatment. Besides stromal tumor infiltrating lymphocytes (sTILs) which have recently reached level 1B evidence as a prognostic marker in triple negative BC, a plethora of methods to assess immune infiltration exists, and it is unclear how these compare to each other and if they can be used interchangeably. Methods Two experienced pathologists scored sTIL, intra-tumoral TIL (itTIL), and 6 immune cell types (CD3+, CD4+, CD8+, CD20+, CD68+, FOXP3+) in the International Cancer Genomics Consortium breast cancer cohort using hematoxylin and eosin-stained (n = 243) and immunohistochemistry-stained tissue microarrays (n = 254) and whole slides (n = 82). The same traits were evaluated using transcriptomic- and methylomic-based deconvolution methods or signatures. Results The concordance correlation coefficient (CCC) between pathologists for sTIL was very good (0.84) and for cell-specific immune infiltrates slightly lower (0.63–0.66). Comparison between tissue microarray and whole slide pathology scores revealed systematically higher values in whole slides (ratio 2.60–5.98). The Spearman correlations between microscopic sTIL and transcriptomic- or methylomic-based assessment of immune infiltrates were highly variable (r = 0.01–0.56). Similar observations were made for cell type-specific quantifications (r = 0.001–0.54). We observed a strong inter-method variability between the omics-derived estimations, which is further cell type dependent. Finally, we demonstrated that most methods more accurately identify highly infiltrated (sTIL ≥ 60%; area under the curve, AUC, 0.64–0.99) as compared to lowly infiltrated tumors (sTIL ≤ 10%; AUC 0.52–0.82). Conclusions There is a lower inter-pathologist concordance for cell-specific quantification as compared to overall infiltration quantification. Microscopic assessments are underestimated when considering small cores (tissue microarray) instead of whole slides. Results further highlight considerable differences between the microscopic-, transcriptomic-, and methylomic-based methods in the assessment of overall and cell-specific immune infiltration in BC. We therefore call for extreme caution when assessing immune infiltrates using current methods and emphasize the need for standardized immune characterization beyond TIL.

Detecting genomic abnormalities in metastatic castration-resistant prostate cancer (mCRPC) may impact clinical treatment. Here, the authors present whole-genome sequencing of metastatic biopsies from 197 mCRPC patients, highlighting the landscape of microsatellite stability, homologous repair deficiency, and other genomic subgroups.

5-Fluorouracil (5-FU) is a commonly used chemo drug to treat cancer patients. Using in vitro 5-FU treated organoids and in vivo 5-FU treated human cancer data, the authors describe a 5-FU mutational signature that is similar to COSMIC signature 17 frequently observed in untreated cancers

Abstract Background Bone morphogenetic proteins (BMPs) have been reported to maintain epithelial integrity and to antagonize the transforming growth factor β (TGFβ)-induced epithelial to mesenchymal transition. The expression of soluble BMP antagonists is dysregulated in cancers and interrupts proper BMP signaling in breast cancer. Methods In this study, we mined the prognostic role of BMP antagonists GREMLIN 1 (GREM1) in primary breast cancer tissues using in-house and publicly available datasets. We determined which cells express GREM1 RNA using in situ hybridization (ISH) on a breast cancer tissue microarray. The effects of Grem1 on the properties of breast cancer cells were assessed by measuring the mesenchymal/stem cell marker expression and functional cell-based assays for stemness and invasion. The role of Grem1 in breast cancer-associated fibroblast (CAF) activation was measured by analyzing the expression of fibroblast markers, phalloidin staining, and collagen contraction assays. The role of Grem1 in CAF-induced breast cancer cell intravasation and extravasation was studied by utilizing xenograft zebrafish breast cancer (co-) injection models. Results Expression analysis of clinical breast cancer datasets revealed that high expression of GREM1 in breast cancer stroma is correlated with a poor prognosis regardless of the molecular subtype. The large majority of human breast cancer cell lines did not express GREM1 in vitro, but breast CAFs did express GREM1 both in vitro and in vivo. Transforming growth factor β (TGFβ) secreted by breast cancer cells, and also inflammatory cytokines, stimulated GREM1 expression in CAFs. Grem1 abrogated bone morphogenetic protein (BMP)/SMAD signaling in breast cancer cells and promoted their mesenchymal phenotype, stemness, and invasion. Moreover, Grem1 production by CAFs strongly promoted the fibrogenic activation of CAFs and promoted breast cancer cell intravasation and extravasation in co-injection xenograft zebrafish models. Conclusions Our results demonstrated that Grem1 is a pivotal factor in the reciprocal interplay between breast cancer cells and CAFs, which promotes cancer cell invasion. Targeting Grem1 could be beneficial in the treatment of breast cancer patients with high Grem1 expression.

The androgen receptor splice variant (AR‐V) 7 in circulating tumor cells (CTCs) is a predictor for resistance to anti‐AR‐targeted treatment, but not to taxane‐based chemotherapy in metastatic castration‐resistant prostate cancer (mCRPC). In this study, we investigated whether the presence of two constitutively active variants (AR‐V3, AR‐V7) and two other conditionally activated variants (AR‐V1, AR‐V9) vs full‐length androgen receptor (AR‐FL) measured in CTCs from patients with mCRPC were associated with outcome to therapy with the taxane cabazitaxel. Blood was collected at baseline and after two cycles of cabazitaxel from 118 mCRPC patients starting cabazitaxel in a prospective phase II trial. CellSearch‐enriched CTCs were enumerated and in parallel characterized for the presence of the AR‐Vs by reverse transcription quantitative polymerase chain reaction. Correlations with CTC and prostate‐specific antigen response to cabazitaxel as well as associations with overall survival (OS) were investigated. All AR‐Vs were frequently present and co‐expressed at frequencies of 31–48% at baseline and at 19–40% after two cycles of cabazitaxel. No specific directions of change in the measured variants were detected between the start of treatment and after two cycles of cabazitaxel. No associations between the presence of AR‐V3 and AR‐V7 and outcome to cabazitaxel were observed. While a reduction in CTCs to

Abstract Background The effective treatment of triple-negative breast cancer (TNBC) remains a profound clinical challenge. Despite frequent epidermal growth factor receptor (EGFR) overexpression and reliance on downstream signalling pathways in TNBC, resistance to EGFR-tyrosine kinase inhibitors (TKIs) remains endemic. Therefore, the identification of targeted agents, which synergise with current therapeutic options, is paramount. Methods Compound-based, high-throughput, proliferation screening was used to profile the response of TNBC cell lines to EGFR-TKIs, western blotting and siRNA transfection being used to examine the effect of inhibitors on EGFR-mediated signal transduction and cellular dependence on such pathways, respectively. A kinase inhibitor combination screen was used to identify compounds that synergised with EGFR-TKIs in TNBC, utilising sulphorhodamine B (SRB) assay as read-out for proliferation. The impact of drug combinations on cell cycle arrest, apoptosis and signal transduction was assessed using flow cytometry, automated live-cell imaging and western blotting, respectively. RNA sequencing was employed to unravel transcriptomic changes elicited by this synergistic combination and to permit identification of the signalling networks most sensitive to co-inhibition. Results We demonstrate that a dual cdc7/CDK9 inhibitor, PHA-767491, synergises with multiple EGFR-TKIs (lapatinib, erlotinib and gefitinib) to overcome resistance to EGFR-targeted therapy in various TNBC cell lines. Combined inhibition of EGFR and cdc7/CDK9 resulted in reduced cell proliferation, accompanied by induction of apoptosis, G2-M cell cycle arrest, inhibition of DNA replication and abrogation of CDK9-mediated transcriptional elongation, in contrast to mono-inhibition. Moreover, high expression of cdc7 and RNA polymerase II Subunit A (POLR2A), the direct target of CDK9, is significantly correlated with poor metastasis-free survival in a cohort of breast cancer patients. RNA sequencing revealed marked downregulation of pathways governing proliferation, transcription and cell survival in TNBC cells treated with the combination of an EGFR-TKI and a dual cdc7/CDK9 inhibitor. A number of genes enriched in these downregulated pathways are associated with poor metastasis-free survival in TNBC. Conclusions Our results highlight that dual inhibition of cdc7 and CDK9 by PHA-767491 is a potential strategy for targeting TNBC resistant to EGFR-TKIs.

Triple-negative breast cancers (TNBC) have enhanced migratory behaviour. Here, the authors perform a phenotypic imaging-based RNAi screen to identify several genes associated with regulation of migratory phenotypes and show that one of the regulators, PRPF4B, mediates metastasis in TNBC in mice.

In cancer, global DNA methylation loss and CpG island hypermethylation are commonly observed. Here, in breast cancer the authors find that hyper-variability of partially methylated domains is the prime source of DNA methylation variation and that these domains fuel CpG island hypermethylation.

The emerging interest in circulating tumor DNA (ctDNA) analyses for clinical trials has necessitated the development of a high‐throughput method for fast, reproducible, and efficient isolation of ctDNA. Currently, the majority of ctDNA studies use the manual QIAamp (QA) platform to isolate DNA from blood. The purpose of this study was to compare two competing automated DNA isolation platforms [Maxwell (MX) and QIAsymphony (QS)] to the current ‘gold standard’ QA to facilitate high‐throughput processing of samples in prospective trials. We obtained blood samples from healthy blood donors and metastatic cancer patients for plasma isolation. Total cell‐free DNA (cfDNA) quantity was assessed by TERT quantitative PCR. Recovery efficiency was investigated by quantitative PCR analysis of spiked‐in synthetic plant DNA. In addition, a β‐actin fragmentation assay was performed to determine the amount of contamination by genomic DNA from lysed leukocytes. ctDNA quality was assessed by digital PCR for somatic variant detection. cfDNA quantity and recovery efficiency were lowest using the MX platform, whereas QA and QS showed a comparable performance. All platforms preferentially isolated small (136 bp) DNA fragments over large (420 and 2000 bp) DNA fragments. Detection of the number variant and wild‐type molecules was most comparable between QA and QS. However, there was no significant difference in variant allele frequency comparing QS and MX to QA. In summary, we show that the QS platform has comparable performance to QA, the ‘gold standard’, and outperformed the MX platform depending on the readout used. We conclude that the QS can replace the more laborious QA platform, especially when high‐throughput cfDNA isolation is needed.

Abstract Background Current normalization methods for RNA-sequencing data allow either for intersample comparison to identify differentially expressed (DE) genes or for intrasample comparison for the discovery and validation of gene signatures. Most studies on optimization of normalization methods typically use simulated data to validate methodologies. We describe a new method, GeTMM, which allows for both inter- and intrasample analyses with the same normalized data set. We used actual (i.e. not simulated) RNA-seq data from 263 colon cancers (no biological replicates) and used the same read count data to compare GeTMM with the most commonly used normalization methods (i.e. TMM (used by edgeR), RLE (used by DESeq2) and TPM) with respect to distributions, effect of RNA quality, subtype-classification, recurrence score, recall of DE genes and correlation to RT-qPCR data. Results We observed a clear benefit for GeTMM and TPM with regard to intrasample comparison while GeTMM performed similar to TMM and RLE normalized data in intersample comparisons. Regarding DE genes, recall was found comparable among the normalization methods, while GeTMM showed the lowest number of false-positive DE genes. Remarkably, we observed limited detrimental effects in samples with low RNA quality. Conclusions We show that GeTMM outperforms established methods with regard to intrasample comparison while performing equivalent with regard to intersample normalization using the same normalized data. These combined properties enhance the general usefulness of RNA-seq but also the comparability to the many array-based gene expression data in the public domain.

Mutations and splice variants in the estrogen receptor (ER) gene, ESR1, may yield endocrine resistance in metastatic breast cancer (MBC) patients. These putative endocrine resistance markers are likely to emerge during treatment, and therefore, its detection in liquid biopsies, such as circulating tumor cells (CTCs) and cell‐free DNA (cfDNA), is of great interest. This research aimed to determine whether ESR1 mutations and splice variants occur more frequently in CTCs of MBC patients progressing on endocrine treatment. In addition, the presence of ESR1 mutations was evaluated in matched cfDNA and compared to CTCs. CellSearch‐enriched CTC fractions (≥5/7.5 mL) of two MBC cohorts were evaluated, namely (a) patients starting first‐line endocrine therapy (n = 43, baseline cohort) and (b) patients progressing on any line of endocrine therapy (n = 40, progressing cohort). ESR1 hotspot mutations (D538G and Y537S/N/C) were evaluated in CTC‐enriched DNA using digital PCR and compared with matched cfDNA (n = 18 baseline cohort; n = 26 progressing cohort). Expression of ESR1 full‐length and 4 of its splice variants (∆5, ∆7, 36 kDa, and 46 kDa) was evaluated in CTC‐enriched mRNA. It was observed that in the CTCs, the ESR1 mutations were not enriched in the progressing cohort (8%), when compared with the baseline cohort (5%) (P = 0.66). In the cfDNA, however, ESR1 mutations were more prevalent in the progressing cohort (42%) than in the baseline cohort (11%) (P = 0.04). Three of the same mutations were observed in both CTCs and cfDNA, 1 mutation in CTCs only, and 11 in cfDNA only. Only the ∆5 ESR1 splice variant was CTC‐specific expressed, but was not enriched in the progressing cohort. In conclusion, sensitivity for detecting ESR1 mutations in CTC‐enriched fractions was lower than for cfDNA. ESR1 mutations detected in cfDNA, rarely present at the start of first‐line endocrine therapy, were enriched at progression, strongly suggesting a role in conferring endocrine resistance in MBC.

A Correction to this paper has been published: https://doi.org/10.1038/s41467-021-23162-4

The identification of transcriptomic alterations of HER2+ ductal carcinoma in situ (DCIS) that are associated with the density of tumor-infiltrating lymphocytes (TILs) could contribute to optimizing choices regarding the potential benefit of immune therapy. We compared the gene expression profile of TIL-poor HER2+ DCIS to that of TIL-rich HER2+ DCIS. Tumor cells from 11 TIL-rich and 12 TIL-poor DCIS cases were micro-dissected for RNA isolation. The Ion AmpliSeq Transcriptome Human Gene Expression Kit was used for RNA sequencing. After normalization, a Mann–Whitney rank sum test was used to analyze differentially expressed genes between TIL-poor and TIL-rich HER2+ DCIS. Whole tissue sections were immunostained for validation of protein expression. We identified a 29-gene expression profile that differentiated TIL-rich from TIL-poor HER2+ DCIS. These genes included CCND3, DUSP10 and RAP1GAP, which were previously described in breast cancer and cancer immunity and were more highly expressed in TIL-rich DCIS. Using immunohistochemistry, we found lower protein expression in TIL-rich DCIS. This suggests regulation of protein expression at the posttranslational level. We identified a gene expression profile of HER2+ DCIS cells that was associated with the density of TILs. This classifier may guide towards more rationalized choices regarding immune-mediated therapy in HER2+ DCIS, such as targeted vaccine therapy.

Abstract The use of blood-circulating cell-free DNA (cfDNA) as ‘liquid-biopsy’ is explored worldwide, with hopes for its potential in providing prognostic or predictive information in cancer treatment. In exploring cfDNA, valuable repositories are biobanks containing material collected over time, however these retrospective cohorts have restrictive resources. In this study, we aimed to detect tumor-specific mutations in only minute amounts of serum-derived cfDNA by using a targeted next generation sequencing (NGS) approach. In a retrospective cohort of ten metastatic breast cancer patients, we profiled DNA from primary tumor tissue (frozen), tumor-adjacent normal tissue (formalin-fixed paraffin embedded), and three consecutive serum samples (frozen). Our presented workflow includes comparisons with matched normal DNA or in silico reference DNA to discriminate germline from somatic variants, validation of variants through the detection in at least two DNA samples of an individual, and the use of public databases on variants. By our workflow, we were able to detect a total of four variants traceable as circulating tumor DNA (ctDNA) in the sera of three of the ten patients.

ABSTRACT APOBEC3B is a single-stranded DNA cytosine deaminase with beneficial innate antiviral functions. However, misregulated APOBEC3B can also be detrimental by inflicting APOBEC signature C-to-T and C-to-G mutations in genomic DNA of multiple cancer types. Polyomavirus and papillomavirus oncoproteins induce APOBEC3B overexpression, perhaps to their own benefit, but little is known about the cellular mechanisms hijacked by these viruses to do so. Here we investigate the molecular mechanism of APOBEC3B upregulation by the polyomavirus large T antigen. First, we demonstrate that the upregulated APOBEC3B enzyme is strongly nuclear and partially localized to virus replication centers. Second, truncated T antigen (truncT) is sufficient for APOBEC3B upregulation, and the RB-interacting motif (LXCXE), but not the p53-binding domain, is required. Third, genetic knockdown of RB1 alone or in combination with RBL1 and/or RBL2 is insufficient to suppress truncT-mediated induction of APOBEC3B. Fourth, CDK4/6 inhibition by palbociclib is also insufficient to suppress truncT-mediated induction of APOBEC3B. Last, global gene expression analyses in a wide range of human cancers show significant associations between expression of APOBEC3B and other genes known to be regulated by the RB/E2F axis. These experiments combine to implicate the RB/E2F axis in promoting APOBEC3B transcription, yet they also suggest that the polyomavirus RB-binding motif has at least one additional function in addition to RB inactivation for triggering APOBEC3B upregulation in virus-infected cells. IMPORTANCE The APOBEC3B DNA cytosine deaminase is overexpressed in many different cancers and correlates with elevated frequencies of C-to-T and C-to-G mutations in 5′-TC motifs, oncogene activation, acquired drug resistance, and poor clinical outcomes. The mechanisms responsible for APOBEC3B overexpression are not fully understood. Here, we show that the polyomavirus truncated T antigen (truncT) triggers APOBEC3B overexpression through its RB-interacting motif, LXCXE, which in turn likely modulates the binding of E2F family transcription factors to promote APOBEC3B expression. This work strengthens the mechanistic linkage between active cell cycling, APOBEC3B overexpression, and cancer mutagenesis. Although this mutational mechanism damages cellular genomes, viruses may leverage it to promote evolution, immune escape, and pathogenesis. The cellular portion of the mechanism may also be relevant to nonviral cancers, where genetic mechanisms often activate the RB/E2F axis and APOBEC3B mutagenesis contributes to tumor evolution.

Recent studies using in depth DNA sequencing techniques led to the identification of cancer driver genes but mainly focused on the effect on their expression. Here, the authors analyse 266 cases of breast cancer and report gene expression signatures associated with the number and character of signature mutations.