Genome-wide cell-free DNA fragmentation in patients with cancer.

Cell-free DNA in the blood provides a non-invasive diagnostic avenue for patients with cancer1. However, characteristics of the origins and molecular features of cell-free DNA are poorly understood. Here we developed an approach to evaluate fragmentation patterns of cell-free DNA across the genome, and found that profiles of healthy individuals reflected nucleosomal patterns of white blood cells, whereas patients with cancer had altered fragmentation profiles. We used this method to analyse the fragmentation profiles of 236 patients with breast, colorectal, lung, ovarian, pancreatic, gastric or bile duct cancer and 245 healthy individuals. A machine learning model that incorporated genome-wide fragmentation features had sensitivities of detection ranging from 57% to more than 99% among the seven cancer types at 98% specificity, with an overall area under the curve value of 0.94. Fragmentation profiles could be used to identify the tissue of origin of the cancers to a limited number of sites in 75% of cases. Combining our approach with mutation-based cell-free DNA analyses detected 91% of patients with cancer. The results of these analyses highlight important properties of cell-free DNA and provide a proof-of-principle approach for the screening, early detection and monitoring of human cancer.

Incidence of late recurrence and second primary cancers 5-10 years after non-metastatic colorectal cancer.

The fraction of patients who are cancer-free survivors 5 years after curative-intended surgery for colorectal cancer (CRC) is increasing, suggesting that extending surveillance beyond 5 years may be indicated. Here we estimate the incidence of late recurrence, metachronous CRC, and second primary cancers 5-10 years postoperative. All patients resected for UICC stage I-III CRC in Denmark through 2004-2013 were identified. Through individual-level linkage of nationwide health registry data, recurrence status was determined using a validated algorithm. Cancer-free survivors 5 years after surgery, were included. Cumulative incidence functions (CIF) of late recurrence, metachronous CRC, and second primary cancer 5-10 years postoperative were constructed. Subdistribution hazards ratios (sHR) were computed using Fine-Gray regression. Among 8883 patients, 370 developed late recurrence (5-10-year CIF = 4.1%, 95%CI: 3.7%-4.6%), 270 metachronous CRC (5-10-year CIF = 3.0%, 95%CI: 2.7%-3.4%), and 635 a second primary cancer (5-10-year CIF = 7.2%, 95%CI: 6.7%-7.7%). The risk of late recurrence was reduced for patients operated in 2009-2013 compared to 2004-2008 (2.9% vs. 5.6%, sHR = 0.52, 95% CI: 0.42-0.65). The risk of metachronous CRC was likewise reduced from 4.1% to 2.1% (sHR = 0.50, 95%CI: 0.39-0.65). While the risk of second primary cancer did not change between 2009-2013 and 2004-2008 (7.1% vs. 7.1%, sHR = 0.98, 95% CI: 0.84-1.15). Using nation-wide 10-year follow-up data, we document that the incidences of late recurrence and metachronous CRC are low and decreasing from 2004 to 2013. Thus, despite increasing numbers of long-term cancer survivors, the data do not advocate for extending CRC-specific surveillance beyond 5 years.

Error-Corrected Deep Targeted Sequencing of Circulating Cell-Free DNA from Colorectal Cancer Patients for Sensitive Detection of Circulating Tumor DNA.

Circulating tumor DNA (ctDNA) is a promising biomarker, reflecting the presence of tumor cells. Sequencing-based detection of ctDNA at low tumor fractions is challenging due to the crude error rate of sequencing. To mitigate this challenge, we developed ultra-deep mutation-integrated sequencing (UMIseq), a fixed-panel deep targeted sequencing approach, which is universally applicable to all colorectal cancer (CRC) patients. UMIseq features UMI-mediated error correction, the exclusion of mutations related to clonal hematopoiesis, a panel of normal samples for error modeling, and signal integration from single-nucleotide variations, insertions, deletions, and phased mutations. UMIseq was trained and independently validated on pre-operative (pre-OP) plasma from CRC patients (n = 364) and healthy individuals (n = 61). UMIseq displayed an area under the curve surpassing 0.95 for allele frequencies (AFs) down to 0.05%. In the training cohort, the pre-OP detection rate reached 80% at 95% specificity, while it was 70% in the validation cohort. UMIseq enabled the detection of AFs down to 0.004%. To assess the potential for detection of residual disease, 26 post-operative plasma samples from stage III CRC patients were analyzed. From this we found that the detection of ctDNA was associated with recurrence. In conclusion, UMIseq demonstrated robust performance with high sensitivity and specificity, enabling the detection of ctDNA at low allele frequencies.

Circulating tumor DNA for prognosis assessment and postoperative management after curative-intent resection of colorectal liver metastases.

The recurrence rate of colorectal liver metastases (CRLM) patients treated with curative intent is above 50%. Standard of care surveillance includes intensive computed tomographic (CT) imaging as well as carcinoembryonic antigen (CEA) measurements. Nonetheless, relapse detection often happens too late to resume curative treatment. This longitudinal cohort study enrolled 115 patients with plasma samples (N = 439) prospectively collected before surgery, postoperatively at day 30 and every third month for up to 3 years. Droplet digital PCR (ddPCR) was used to monitor serial plasma samples for somatic mutations. Assessment of ctDNA status either immediately after surgery, or serially during surveillance, stratified the patients into groups of high and low recurrence risk (hazard ratio [HR], 7.6; 95% CI, 3.0-19.7; P < .0001; and HR, 4.3; 95% CI, 2.3-8.1; P < .0001, respectively). The positive predictive value (PPV) of ctDNA was 100% in all postoperative analyses. In multivariable analyses, postoperative ctDNA status was the only consistently significant risk marker associated with relapse (P < .0001). Indeterminate CT findings were observed for 30.8% (21/68) of patients. All patients (9/21) that were ctDNA positive at the time of the indeterminate CT scan later relapsed, contrasting 42.6% (5/12) of those ctDNA negative (P = .0046). Recurrence diagnoses in patients with indeterminate CT findings were delayed (median 2.8 months, P < .0001). ctDNA status is strongly associated with detection of minimal residual disease and early detection of relapse. Furthermore, ctDNA status can potentially contribute to clinical decision-making in case of indeterminate CT findings, reducing time-to-intervention.

ctDNA for Risk of Recurrence Assessment in Patients Treated with Neoadjuvant Treatment: A Systematic Review and Meta-analysis.

BACKGROUND: We wanted to investigate the association between circulating tumor DNA (ctDNA) detection at baseline, during and after neoadjuvant treatment, after surgery, and recurrence, in patients with nonmetastatic cancer. PATIENTS AND METHODS: In this systematic review and meta-analysis, we included studies that investigated patients undergoing neoadjuvant treatment for nonmetastatic cancer and provided recurrence indices stratified for ctDNA status at the following timepoints: baseline, during treatment, posttreatment, and postsurgery. Study quality was reported with the Newcastle-Ottawa scale, REMARK checklist, and GRADE approach. PubMed, Embase, Cochrane Library, and Web of Science were our data sources (inception to 3 June 2021). The main outcome was risk of recurrence. RESULTS: We identified ten studies including 727 patients with rectal, breast, gastric, and bladder cancer. All studies reported posttreatment ctDNA analysis, while seven, four, and six reported baseline, during treatment, and postsurgery ctDNA analysis, respectively. ctDNA detection was associated to recurrence across all timepoints [baseline: risk ratio (RR) 2.86, 95% confidence interval (CI) 1.33-6.14, during treatment: RR 3.81, 95% CI 2.09-6.92, posttreatment: RR 4.29, 95% CI 2.79-6.60, postsurgery: RR 8.03, 95% CI 3.16-20.43]. Heterogeneity was low to moderate. CONCLUSIONS: This meta-analysis of observational studies found that ctDNA detection in patients undergoing neoadjuvant treatment for nonmetastatic cancer was associated with recurrence. A stronger association was evident in posttreatment and postsurgery timepoints. However, some studies reported low negative predictive value (NPV) of pathological complete response, showing that ctDNA-detection-guided escalation and de-escalation studies following neoadjuvant treatment regimens are needed before its role as a treatment guidance can be affirmed.

Age-stratified reference intervals unlock the clinical potential of circulating cell-free DNA as a biomarker of poor outcome for healthy individuals and patients with colorectal cancer.

Circulating cell-free DNA (cfDNA) has spurred much interest as a biomarker in oncology. However, inter- and intra-individual cfDNA levels vary greatly. Consequently, in order to base clinical decisions on cfDNA measurements, normal reference intervals are essential to avoid that ordinary variation is confused with clinically relevant change. The lack of reference intervals may potentially explain the ambiguous results reported in the field. Our study aimed to establish reference intervals and to evaluate the association between cfDNA and demographic and clinical variables, including colorectal cancer (CRC). Plasma samples and clinical data from 2817 subjects were collected including 1930 noncancer individuals and 887 CRC patients. cfDNA was measured using droplet digital polymerase chain reaction (PCR). The large cohort combined with robust cfDNA quantification enabled establishment of reference intervals (<67 years: 775-4860 copies/mL; ≥67 years: 807-6561 copies/mL). A cfDNA level above the age-stratified 90% percentile was prognostic of reduced survival in both noncancer individuals and CRC patients, with HR values of 2.56 and 2.01, respectively. Moreover, cfDNA levels increased significantly with age, elevated BMI and chronic diseases. In CRC, the cfDNA level was increased for Stage IV, but not Stage I to Stage III cancer. In summary, the use of reference intervals revealed that high cfDNA levels were predictive of shorter survival in both noncancer individuals and CRC patients, and that CRC development did not affect the cfDNA level until metastatic dissemination. Furthermore, cfDNA levels were impacted by age and chronic diseases. Conclusively, our study presents reference intervals that will help pave the way for clinical utilization of cfDNA.

Validation of computational determination of microsatellite status using whole exome sequencing data from colorectal cancer patients.

BACKGROUND: Microsatellite instability (MSI), resulting from a defective mismatch repair system, occurs in approximately 15% of sporadic colorectal cancers (CRC). Since MSI is associated with a poor response to 5-fluorouracile based chemotherapy and is a positive predictive marker of immunotherapy, it is routine practice to evaluate the MSI status of resected tumors in CRC patients. MSIsensor is a novel computational tool for determining MSI status using Next Generation Sequencing. However, it is not widely used in the clinic and has not been independently validated in exome data from CRC. To facilitate clinical implementation of computational determination of MSI status, we compared MSIsensor to current gold standard methods for MSI testing. METHODS: MSI status was determined for 130 CRC patients (UICC stage I-IV) using immunohistochemistry, PCR based microsatellite stability testing and by applying MSIsensor to exome sequenced tumors and paired germline DNA. Furthermore, we investigated correlation between MSI status, mutational load and mutational signatures. RESULTS: Eighteen out of 130 (13.8%) patients were microsatellite instable. We found a 100% agreement between MSIsensor and gold standard methods for MSI testing. All MSI tumors were hypermutated. In addition, two microsatellite stable (MSS) tumors were hypermutated, which was explained by a dominant POLE signature and pathogenic POLE mutations (p.Pro286Arg and p.Ser459Phe). CONCLUSION: MSIsensor is a robust tool, which can be used to determine MSI status of tumor samples from exome sequenced CRC patients.

The potential role of Alu Y in the development of resistance to SN38 (Irinotecan) or oxaliplatin in colorectal cancer.

BACKGROUND: Irinotecan (SN38) and oxaliplatin are chemotherapeutic agents used in the treatment of colorectal cancer. However, the frequent development of resistance to these drugs represents a considerable challenge in the clinic. Alus as retrotransposons comprise 11% of the human genome. Genomic toxicity induced by carcinogens or drugs can reactivate Alus by altering DNA methylation. Whether or not reactivation of Alus occurs in SN38 and oxaliplatin resistance remains unknown. RESULTS: We applied reduced representation bisulfite sequencing (RRBS) to investigate the DNA methylome in SN38 or oxaliplatin resistant colorectal cancer cell line models. Moreover, we extended the RRBS analysis to tumor tissue from 14 patients with colorectal cancer who either did or did not benefit from capecitabine + oxaliplatin treatment. For the clinical samples, we applied a concept of 'DNA methylation entropy' to estimate the diversity of DNA methylation states of the identified resistance phenotype-associated methylation loci observed in the cell line models. We identified different loci being characteristic for the different resistant cell lines. Interestingly, 53% of the identified loci were Alu sequences- especially the Alu Y subfamily. Furthermore, we identified an enrichment of Alu Y sequences that likely results from increased integration of new copies of Alu Y sequence in the drug-resistant cell lines. In the clinical samples, SOX1 and other SOX gene family members were shown to display variable DNA methylation states in their gene regions. The Alu Y sequences showed remarkable variation in DNA methylation states across the clinical samples. CONCLUSION: Our findings imply a crucial role of Alu Y in colorectal cancer drug resistance. Our study underscores the complexity of colorectal cancer aggravated by mobility of Alu elements and stresses the importance of personalized strategies, using a systematic and dynamic view, for effective cancer therapy.

Determinants of recurrence after intended curative resection for colorectal cancer.

Despite intended curative resection, colorectal cancer will recur in ∼45% of the patients. Results of meta-analyses conclude that frequent follow-up does not lead to early detection of recurrence, but improves overall survival. The present literature shows that several factors play important roles in development of recurrence. It is well established that emergency surgery is a major determinant of recurrence. Moreover, anastomotic leakages, postoperative bacterial infections, and blood transfusions increase the recurrence rates although the exact mechanisms still remain obscure. From pathology studies it has been shown that tumors behave differently depending on their location and recur more often when micrometastases are present in lymph nodes and around vessels and nerves. K-ras mutations, microsatellite instability, and mismatch repair genes have also been shown to be important in relation with recurrences, and tumors appear to have different mutations depending on their location. Patients with stage II or III disease are often treated with adjuvant chemotherapy despite the fact that the treatments are far from efficient among all patients, who are at risk of recurrence. Studies are now being presented identifying subgroups, in which the therapy is inefficient. Unfortunately, only few of these facts are implemented in the present follow-up programs. Therefore, further research is urgently needed to verify which of the well-known parameters as well as new parameters that must be added to the current follow-up programs to identify patients at risk of recurrence.

Incidence of Recurrence and Time to Recurrence in Stage I to III Colorectal Cancer: A Nationwide Danish Cohort Study.

Importance: Management of colorectal cancer (CRC) has been updated continuously over the past 2 decades. While the combination of these initiatives has had implications for improved survival, the implications for rates of recurrence remain unexplored. Objective: To ascertain the rates of recurrence and describe time to recurrence within 5 years of surgery with curative intent for stages I to III CRC. Design, Setting, and Participants: This cohort study used the Danish Colorectal Cancer Group Database to identify patients with Union for International Cancer Control (UICC) stages I to III CRC who underwent primary surgery between January 1, 2004, and December 31, 2019. They were followed up until recurrence (event), death (competing event), diagnosis of a second cancer (competing event), emigration (censoring event), 5 years postoperatively (censoring event), or January 1, 2023 (censoring event), whichever came first. Recurrence status was ascertained through individual-level linked data from the Danish Cancer Registry, Danish National Patient Registry, and Danish Pathology Registry using a validated algorithm. Data were analyzed from January 1 to August 8, 2023. Exposure: Primary surgery performed during 3 calendar periods (2004-2008, 2009-2013, and 2014-2019) stratified by tumor site (colon or rectum) and UICC stage (I, II, and III). Main Outcomes and Measures: Stage-specific 5-year recurrence reported as the cumulative incidence function (CIF) of recurrence, the association between calendar period of primary surgery and recurrence risk reported as subdistribution hazard ratios (sHRs), and the time from surgery to recurrence. Results: Of the 34 166 patients with UICC stages I to III CRC (median [IQR] age, 70 [62-77] years); 18 552 males [54.3%]) included in the study, 7027 developed recurrence within 5 years after the primary surgery. For colon cancer, the 5-year CIF of recurrence decreased over the 3 calendar periods from 16.3% to 6.8% for UICC stage I, from 21.9% to 11.6% for UICC stage II, and from 35.3% to 24.6% for UICC stage III colon cancer. For rectal cancer, the 5-year CIF decreased over the 3 periods from 19.9% to 9.5% for stage I, from 25.8% to 18.4% for stage II, and from 38.7% to 28.8% for stage III disease. Patients with stage III disease had a shorter time from surgery to recurrence compared with those with stage I disease (time ratio stage III vs stage I = 0.30; 95% CI, 0.28-0.32). Cancers detected through screening were associated with lower stage-adjusted risks of recurrence (sHR, 0.81; 95% CI, 0.73-0.91) compared with cancers not detected through screening. Conclusions and Relevance: In this cohort of patients with CRC, the risk of recurrence decreased in patients with stages I to III disease during the study period. Cancer detection by screening was associated with an even lower risk of recurrence. Time to recurrence differed according to UICC stage. Because the risk of recurrence was so low in selected patient groups, future research is warranted to explore risk-stratified surveillance protocols in patients with CRC.

Whole-genome Mutational Analysis for Tumor-informed Detection of Circulating Tumor DNA in Patients with Urothelial Carcinoma.

BACKGROUND AND OBJECTIVE: Circulating tumor DNA (ctDNA) can be used for sensitive detection of minimal residual disease (MRD). However, the probability of detecting ctDNA in settings of low tumor burden is limited by the number of mutations analyzed and the plasma volume available. We used a whole-genome sequencing (WGS) approach for ctDNA detection in patients with urothelial carcinoma. METHODS: We used a tumor-informed WGS approach for ctDNA-based detection of MRD and evaluation of treatment responses. We analyzed 916 longitudinally collected plasma samples from 112 patients with localized muscle-invasive bladder cancer who received neoadjuvant chemotherapy (NAC) before radical cystectomy. Recurrence-free survival (primary endpoint), overall survival, and ctDNA dynamics during NAC were assessed. KEY FINDINGS AND LIMITATIONS: We found that WGS-based ctDNA detection is prognostic for patient outcomes with a median lead time of 131 d over radiographic imaging. WGS-based ctDNA assessment after radical cystectomy identified recurrence with sensitivity of 91% and specificity of 92%. In addition, genomic characterization of post-treatment plasma samples with a high ctDNA level revealed acquisition of platinum therapy-associated mutational signatures and copy number variations not present in the primary tumors. The sequencing depth is a limitation for studying tumor evolution. CONCLUSIONS AND CLINICAL IMPLICATIONS: Our results support the use of WGS for ultrasensitive ctDNA detection and highlight the possibility of plasma-based tracking of tumor evolution. WGS-based ctDNA detection represents a promising option for clinical use owing to the low volume of plasma needed and the ease of performing WGS, eliminating the need for personalized assay design.

Circulating U2 small nuclear RNA fragments as a novel diagnostic biomarker for pancreatic and colorectal adenocarcinoma.

Improved non-invasive strategies for early cancer detection are urgently needed to reduce morbidity and mortality. Non-coding RNAs, such as microRNAs and small nucleolar RNAs, have been proposed as biomarkers for non-invasive cancer diagnosis. Analyzing serum derived from nude mice implanted with primary human pancreatic ductal adenocarcinoma (PDAC), we identified 15 diagnostic microRNA candidates. Of those miR-1246 was selected based on its high abundance in serum of tumor carrying mice. Subsequently, we noted a cross reactivity of the established miR-1246 assays with RNA fragments derived from U2 small nuclear RNA (RNU2-1). Importantly, we found that the assay signal discriminating tumor from controls was derived from U2 small nuclear RNA (snRNA) fragments (RNU2-1f) and not from miR-1246. In addition, we observed a remarkable stability of RNU2-1f in serum and provide experimental evidence that hsa-miR-1246 is likely a pseudo microRNA. In a next step, RNU2-1f was measured by qRT-PCR and normalized to cel-54 in 191 serum/plasma samples from PDAC and colorectal carcinoma (CRC) patients. In comparison to 129 controls, we were able to classify samples as cancerous with a sensitivity and specificity of 97.7% [95% CI = (87.7, 99.9)] and 90.6% [95% CI = (80.7, 96.5)], respectively [area under the ROC curve 0.972]. Of note, patients with CRC were detected with our assay as early as UICC Stage II with a sensitivity of 81%. In conclusion, this is the first report showing that fragments of U2 snRNA are highly stable in serum and plasma and may serve as novel diagnostic biomarker for PDAC and CRC for future prospective screening studies.

Cell-free DNA approaches for cancer early detection and interception.

Rapid advancements in the area of early cancer detection have brought us closer to achieving the goals of finding cancer early enough to treat or cure it, while avoiding harms of overdiagnosis. We evaluate progress in the development of early cancer detection tests in the context of the current principles for cancer screening. We review cell-free DNA (cfDNA)-based approaches using mutations, methylation, or fragmentomes for early cancer detection. Lastly, we discuss the challenges in demonstrating clinical utility of these tests before integration into routine clinical care.

Identifying Recurrences Among Non-Metastatic Colorectal Cancer Patients Using National Health Data Registries: Validation and Optimization of a Registry-Based Algorithm in a Modern Danish Cohort.

Purpose: Colorectal cancer (CRC) recurrence is not routinely recorded in Danish health data registries. Here, we aimed to revalidate a registry-based algorithm to identify recurrences in a contemporary cohort and to investigate the accuracy of estimating the time to recurrence (TTR). Patients and Methods: We ascertained data on 1129 patients operated for UICC TNM stage I-III CRC during 2012-2017 registered in the CRC biobank at the Department of Molecular Medicine, Aarhus University Hospital, Denmark. Individual-level data were linked with data from the Danish Colorectal Cancer Group database, Danish Cancer Registry, Danish National Registry of Patients, and Danish Pathology Registry. The algorithm identified recurrence based on diagnosis codes of local recurrence or metastases, the receipt of chemotherapy, or a pathological tissue assessment code of recurrence more than 180 days after CRC surgery. A subgroup was selected for validation of the algorithm using medical record reviews as a reference standard. Results: We found a 3-year cumulative recurrence rate of 20% (95% CI: 17-22%). Manual medical record review identified 80 recurrences in the validation cohort of 522 patients. The algorithm detected recurrence with 94% sensitivity (75/80; 95% CI: 86-98%) and 98% specificity (431/442; 95% CI: 96-99%). The positive and negative predictive values of the algorithm were 87% (95% CI: 78-93%) and 99% (95% CI: 97-100%), respectively. The median difference in TTR (TTRMedical_chart-TTRalgorithm) was -8 days (IQR: -21 to +3 days). Restricting the algorithm to chemotherapy codes from oncology departments increased the positive predictive value from 87% to 94% without changing the negative predictive value (99%). Conclusion: The algorithm detected recurrence and TTR with high precision in this contemporary cohort. Restriction to chemotherapy codes from oncology departments using department classifications improves the algorithm. The algorithm is suitable for use in future observational studies.

Impact of Whole Genome Doubling on Detection of Circulating Tumor DNA in Colorectal Cancer.

OBJECTIVE: Circulating tumor DNA (ctDNA) is a candidate biomarker of cancer with practice-changing potential in the detection of both early and residual disease. Disease stage and tumor size affect the probability of ctDNA detection, whereas little is known about the influence of other tumor characteristics on ctDNA detection. This study investigates the impact of tumor cell whole-genome doubling (WGD) on the detection of ctDNA in plasma collected preoperatively from newly diagnosed colorectal cancer (CRC) patients. METHODS: WGD was estimated from copy numbers derived from whole-exome sequencing (WES) data of matched tumor and normal DNA from 833 Danish CRC patients. To explore if tumor WGD status impacts ctDNA detection, we applied tumor-informed ctDNA analysis to preoperative plasma samples from all patients. RESULTS: Patients with WGD+ tumors had 53% increased odds of being ctDNA positive (OR = 1.53, 95%CI: 1.12-2.09). After stratification for UICC stage, the association persisted for Stage I (OR = 2.44, 95%CI: 1.22-5.03) and Stage II (OR = 1.76, 95%CI: 1.11-2.81) but not for Stage III (OR = 0.83, 95%CI: 0.44-1.53) patients. CONCLUSION: The presence of WGD significantly increases the probability of detecting ctDNA, particularly for early-stage disease. In patients with more advanced disease, the benefit of WGD on ctDNA detection is less pronounced, consistent with increased DNA shedding from these tumors, making ctDNA detection less dependent on the amount of ctDNA released per tumor cell.

MSIMEP: Predicting microsatellite instability from microarray DNA methylation tumor profiles.

Deficiency in DNA MMR activity results in tumors with a hypermutator phenotype, termed microsatellite instability (MSI). Beyond its utility in Lynch syndrome screening algorithms, today MSI has gained importance as predictive biomarker for various anti-PD-1 therapies across many different tumor types. Over the past years, many computational methods have emerged to infer MSI using either DNA- or RNA-based approaches. Considering this together with the fact that MSI-high tumors frequently exhibit a hypermethylated phenotype, herein we developed and validated MSIMEP, a computational tool for predicting MSI status from microarray DNA methylation tumor profiles of colorectal cancer samples. We demonstrated that MSIMEP optimized and reduced models have high performance in predicting MSI in different colorectal cancer cohorts. Moreover, we tested its consistency in other tumor types with high prevalence of MSI such as gastric and endometrial cancers. Finally, we demonstrated better performance of both MSIMEP models vis-à-vis a MLH1 promoter methylation-based one in colorectal cancer.

Transposon-activated POU5F1B promotes colorectal cancer growth and metastasis.

The treatment of colorectal cancer (CRC) is an unmet medical need in absence of early diagnosis. Here, upon characterizing cancer-specific transposable element-driven transpochimeric gene transcripts (TcGTs) produced by this tumor in the SYSCOL cohort, we find that expression of the hominid-restricted retrogene POU5F1B through aberrant activation of a primate-specific endogenous retroviral promoter is a strong negative prognostic biomarker. Correlating this observation, we demonstrate that POU5F1B fosters the proliferation and metastatic potential of CRC cells. We further determine that POU5F1B, in spite of its phylogenetic relationship with the POU5F1/OCT4 transcription factor, is a membrane-enriched protein that associates with protein kinases and known targets or interactors as well as with cytoskeleton-related molecules, and induces intracellular signaling events and the release of trans-acting factors involved in cell growth and cell adhesion. As POU5F1B is an apparently non-essential gene only lowly expressed in normal tissues, and as POU5F1B-containing TcGTs are detected in other tumors besides CRC, our data provide interesting leads for the development of cancer therapies.

Unsupervised detection of fragment length signatures of circulating tumor DNA using non-negative matrix factorization.

Sequencing of cell-free DNA (cfDNA) is currently being used to detect cancer by searching both for mutational and non-mutational alterations. Recent work has shown that the length distribution of cfDNA fragments from a cancer patient can inform tumor load and type. Here, we propose non-negative matrix factorization (NMF) of fragment length distributions as a novel and completely unsupervised method for studying fragment length patterns in cfDNA. Using shallow whole-genome sequencing (sWGS) of cfDNA from a cohort of patients with metastatic castration-resistant prostate cancer (mCRPC), we demonstrate how NMF accurately infers the true tumor fragment length distribution as an NMF component - and that the sample weights of this component correlate with ctDNA levels (r=0.75). We further demonstrate how using several NMF components enables accurate cancer detection on data from various early stage cancers (AUC = 0.96). Finally, we show that NMF, when applied across genomic regions, can be used to discover fragment length signatures associated with open chromatin.