Modeling Personalized Adjuvant TreaTment in EaRly stage coloN cancer (PATTERN)

Aim: To develop a decision model for the population-level evaluation of strategies to improve the selection of stage II colon cancer (CC) patients who benefit from adjuvant chemotherapy. Methods: A Markov cohort model with a one-month cycle length and a lifelong time horizon was developed. Five health states were included; diagnosis, 90-day mortality, death other causes, recurrence and CC death. Data from the Netherlands Cancer Registry were used to parameterize the model. Transition probabilities were estimated using parametric survival models including relevant clinical and pathological covariates. Subsequently, biomarker status was implemented using external data. Treatment effect was incorporated using pooled trial data. Model development, data sources used, parameter estimation, and internal and external validation are described in detail. To illustrate the use of the model, three example strategies were evaluated in which allocation of treatment was based on (A) 100% adherence to the Dutch guidelines, (B) observed adherence to guideline recommendations and (C) a biomarker-driven strategy. Results: Overall, the model showed good internal and external validity. Age, tumor growth, tumor sidedness, evaluated lymph nodes, and biomarker status were included as covariates. For the example strategies, the model predicted 83, 87 and 77 CC deaths after 5 years in a cohort of 1000 patients for strategies A, B and C, respectively. Conclusion: This model can be used to evaluate strategies for the allocation of adjuvant chemotherapy in stage II CC patients. In future studies, the model will be used to estimate population-level long-term health gain and cost-effectiveness of biomarker-based selection strategies

Modeling Personalized Adjuvant TreaTment in EaRly stage coloN cancer (PATTERN)

Aim To develop a decision model for the population-level evaluation of strategies to improve the selection of stage II colon cancer (CC) patients who benefit from adjuvant chemotherapy. Methods A Markov cohort model with a one-month cycle length and a lifelong time horizon was developed. Five health states were included; diagnosis, 90-day mortality, death other causes, recurrence and CC death. Data from the Netherlands Cancer Registry were used to parameterize the model. Transition probabilities were estimated using parametric survival models including relevant clinical and pathological covariates. Subsequently, biomarker status was implemented using external data. Treatment effect was incorporated using pooled trial data. Model development, data sources used, parameter estimation, and internal and external validation are described in detail. To illustrate the use of the model, three example strategies were evaluated in which allocation of treatment was based on (A) 100% adherence to the Dutch guidelines, (B) observed adherence to guideline recommendations and (C) a biomarker-driven strategy. Results Overall, the model showed good internal and external validity. Age, tumor growth, tumor sidedness, evaluated lymph nodes, and biomarker status were included as covariates. For the example strategies, the model predicted 83, 87 and 77 CC deaths after 5 years in a cohort of 1000 patients for strategies A, B and C, respectively. Conclusion This model can be used to evaluate strategies for the allocation of adjuvant chemotherapy in stage II CC patients. In future studies, the model will be used to estimate population-level long-term health gain and cost-effectiveness of biomarker-based selection strategies.Financial support for this study was provided by a grant from ZonMw (Grant number: 848015007). ZonMw had no role in designing the study, interpreting the data, writing the manuscript, and publishing the report

Overexpression of the miR-17-92 cluster in colorectal adenoma organoids causes a carcinoma-like gene expression signature

Gain of chromosome arm 13q is one of the most prevalent DNA copy number alterations associated with colorectal adenoma-tocarcinoma progression. The oncogenic miR-17-92 cluster, located at 13q, was found to be overexpressed in colorectal cancer and in adenomas harboring 13q gain. However, to what extent overexpression of this group of microRNAs actually drives progression to cancer remains to be resolved. Therefore, we aimed to clarify the role of miR-17-92 cluster in the progression from colorectal adenoma to carcinoma.The miR-17-92 cluster was overexpressed in human colorectal adenoma organoids without 13q gain and downstream effects on mRNA expression were investigated, along with functional consequences in vitro and in vivo.Comparison of mRNA sequencing results of organoids overexpressing miR-17-92 and cultures transduced with control vector revealed a miR-17-92 expression signature. This signature appeared to be enriched in an independent series of colorectal cancers and adenomas with 13q gain, confirming that miR-17-92 expression is associated with malignant progression. However, tumor-associated characteristics, such as increased proliferation rate, were not observed in miR-17-92 overexpressing adenoma organoids in vitro. In addition, subcutaneous injection of these organoids in immunodeficient mice was insufficient to cause tumor outgrowth.In conclusion, this study showed that miR-17-92 expression contributes to 13q gain-associated adenoma-to-carcinoma progression, however, this is insufficient to cause malignancy.Cellular mechanisms in basic and clinical gastroenterology and hepatolog

Circulating tumor DNA guided adjuvant chemotherapy in stage II colon cancer (MEDOCC-CrEATE):study protocol for a trial within a cohort study

BACKGROUND: Accurate detection of patients with minimal residual disease (MRD) after surgery for stage II colon cancer (CC) remains an urgent unmet clinical need to improve selection of patients who might benefit form adjuvant chemotherapy (ACT). Presence of circulating tumor DNA (ctDNA) is indicative for MRD and has high predictive value for recurrent disease. The MEDOCC-CrEATE trial investigates how many stage II CC patients with detectable ctDNA after surgery will accept ACT and whether ACT reduces the risk of recurrence in these patients. METHODS/DESIGN: MEDOCC-CrEATE follows the 'trial within cohorts' (TwiCs) design. Patients with colorectal cancer (CRC) are included in the Prospective Dutch ColoRectal Cancer cohort (PLCRC) and give informed consent for collection of clinical data, tissue and blood samples, and consent for future randomization. MEDOCC-CrEATE is a subcohort within PLCRC consisting of 1320 stage II CC patients without indication for ACT according to current guidelines, who are randomized 1:1 into an experimental and a control arm. In the experimental arm, post-surgery blood samples and tissue are analyzed for tissue-informed detection of plasma ctDNA, using the PGDx elio™ platform. Patients with detectable ctDNA will be offered ACT consisting of 8 cycles of capecitabine plus oxaliplatin while patients without detectable ctDNA and patients in the control group will standard follow-up according to guideline. The primary endpoint is the proportion of patients receiving ACT when ctDNA is detectable after resection. The main secondary outcome is 2-year recurrence rate (RR), but also includes 5-year RR, disease free survival, overall survival, time to recurrence, quality of life and cost-effectiveness. Data will be analyzed by intention to treat. DISCUSSION: The MEDOCC-CrEATE trial will provide insight into the willingness of stage II CC patients to be treated with ACT guided by ctDNA biomarker testing and whether ACT will prevent recurrences in a high-risk population. Use of the TwiCs design provides the opportunity to randomize patients before ctDNA measurement, avoiding ethical dilemmas of ctDNA status disclosure in the control group. TRIAL REGISTRATION: Netherlands Trial Register: NL6281/NTR6455 . Registered 18 May 2017, https://www.trialregister.nl/trial/6281

Proteins in stool as biomarkers for non-invasive detection of colorectal adenomas with high risk of progression

Screening to detect colorectal cancer (CRC) in an early or premalignant state is an effective method to reduce CRC mortality rates. Current stool-based screening tests, e.g. fecal immunochemical test (FIT), have a suboptimal sensitivity for colorectal adenomas and difficulty distinguishing adenomas at high risk of progressing to cancer from those at lower risk. We aimed to identify stool protein biomarker panels that can be used for the early detection of high-risk adenomas and CRC. Proteomics data (LC–MS/MS) were collected on stool samples from adenoma (n = 71) and CRC patients (n = 81) as well as controls (n = 129). Colorectal adenoma tissue samples were characterized by low-coverage whole-genome sequencing to determine their risk of progression based on specific DNA copy number changes. Proteomics data were used for logistic regression modeling to establish protein biomarker panels. In total, 15 of the adenomas (15.8%) were defined as high risk of progressing to cancer. A protein panel, consisting of haptoglobin (Hp), LAMP1, SYNE2, and ANXA6, was identified for the detection of high-risk adenomas (sensitivity of 53% at specificity of 95%). Two panels, one consisting of Hp and LRG1 and one of Hp, LRG1, RBP4, and FN1, were identified for high-risk adenomas and CRCs detection (sensitivity of 66% and 62%, respectively, at specificity of 95%). Validation of Hp as a biomarker for high-risk adenomas and CRCs was performed using an antibody-based assay in FIT samples from a subset of individuals from the discovery series (n = 158) and an independent validation series (n = 795). Hp protein was significantly more abundant in high-risk adenoma FIT samples compared to controls in the discovery (p = 0.036) and the validation series (p = 9e-5). We conclude that Hp, LAMP1, SYNE2, LRG1, RBP4, FN1, and ANXA6 may be of value as stool biomarkers for early detection of high-risk adenomas and CRCs

Comparison of NTRK fusion detection methods in microsatellite-instability-high metastatic colorectal cancer

Tropomyosin receptor kinase (TRK) inhibitors have been approved for metastatic solid tumors harboring NTRK fusions, but the detection of NTRK fusions is challenging. International guidelines recommend pan-TRK immunohistochemistry (IHC) screening followed by next generation sequencing (NGS) in tumor types with low prevalence of NTRK fusions, including metastatic colorectal cancer (mCRC). RNA-based NGS is preferred, but is expensive, time-consuming, and extracting good-quality RNA from FFPE tissue is challenging. Alternatives in daily clinical practice are warranted. We assessed the diagnostic performance of RNA-NGS, FFPE-targeted locus capture (FFPE-TLC), fluorescence in situ hybridization (FISH), and the 5'/3' imbalance quantitative RT-PCR (qRT-PCR) after IHC screening in 268 patients with microsatellite-instability-high mCRC, the subgroup in which NTRK fusions are most prevalent (1-5%). A consensus result was determined after review of all assay results. In 16 IHC positive tumors, 10 NTRK fusions were detected. In 33 IHC negative samples, no additional transcribed NTRK fusions were found, underscoring the high sensitivity of IHC. Sensitivity of RNA-NGS, FFPE-TLC, FISH, and qRT-PCR was 90%, 90%, 78%, and 100%, respectively. Specificity was 100% for all assays. Robustness, defined as the percentage of samples that provided an interpretable result in the first run, was 100% for FFPE-TLC, yet more limited for RNA-NGS (85%), FISH (70%), and qRT-PCR (70%). Overall, we do not recommend FISH for the detection of NTRK fusions in mCRC due to its low sensitivity and limited robustness. We conclude that RNA-NGS, FFPE-TLC, and qRT-PCR are appropriate assays for NTRK fusion detection, after enrichment with pan-TRK IHC, in routine clinical practice

Presence of HIF-1 and related genes in normal mucosa, adenomas and carcinomas of the colorectum

Expression of the transcription factor hypoxia-inducible factor 1 (HIF-1), which plays a key role in cellular adaptation to hypoxia, was investigated in normal colorectal mucosa (ten), adenomas (61), and carcinomas (23). Tissue samples were analyzed for HIF-1α, its upstream regulators, von Hippel–Lindau factor, AKT, and mammalian target of rapamycin (mTOR) and its downstream targets glucose transporter 1 (GLUT1), carbonic anhydrase IX, stromal-cell-derived factor 1 (SDF-1) by immunohistochemistry. In normal colorectal mucosa, HIF-1α was observed in almost all nuclei of surface epithelial cells, probably secondary to a gradient of oxygenation, as indicated by pimonidazole staining. The same staining pattern was present in 87% of adenomas. In carcinomas, HIF-1α was present predominantly around areas of necrosis (78%). Active AKT and mTOR, were present in all adenomas, carcinomas, and in normal colorectal mucosa. GLUT1 and SDF-1 were present in the normal surface epithelium of all adenoma cases, whereas in the carcinoma GLUT1 was located around necrotic regions and SDF-1 was present in all epithelial cells. In conclusion, HIF-1α appears to be physiologically expressed in the upper part of the colorectal mucosa. The present observations support that upregulation of HIF-1α and its downstream targets GLUT1 and SDF-1 in colorectal adenomas and carcinomas may be due to hypoxia, in close interaction with an active phosphatidylinositol 3-kinases–AKT–mTOR pathway

Synchronizing Allelic Effects of Opposing Quantitative Trait Loci Confirmed a Major Epistatic Interaction Affecting Acute Lung Injury Survival in Mice

Increased oxygen (O2) levels help manage severely injured patients, but too much for too long can cause acute lung injury (ALI), acute respiratory distress syndrome (ARDS) and even death. In fact, continuous hyperoxia has become a prototype in rodents to mimic salient clinical and pathological characteristics of ALI/ARDS. To identify genes affecting hyperoxia-induced ALI (HALI), we previously established a mouse model of differential susceptibility. Genetic analysis of backcross and F2 populations derived from sensitive (C57BL/6J; B) and resistant (129X1/SvJ; X1) inbred strains identified five quantitative trait loci (QTLs; Shali1-5) linked to HALI survival time. Interestingly, analysis of these recombinant populations supported opposite within-strain effects on survival for the two major-effect QTLs. Whereas Shali1 alleles imparted the expected survival time effects (i.e., X1 alleles increased HALI resistance and B alleles increased sensitivity), the allelic effects of Shali2 were reversed (i.e., X1 alleles increased HALI sensitivity and B alleles increased resistance). For in vivo validation of these inverse allelic effects, we constructed reciprocal congenic lines to synchronize the sensitivity or resistance alleles of Shali1 and Shali2 within the same strain. Specifically, B-derived Shali1 or Shali2 QTL regions were transferred to X1 mice and X1-derived QTL segments were transferred to B mice. Our previous QTL results predicted that substituting Shali1 B alleles onto the resistant X1 background would add sensitivity. Surprisingly, not only were these mice more sensitive than the resistant X1 strain, they were more sensitive than the sensitive B strain. In stark contrast, substituting the Shali2 interval from the sensitive B strain onto the X1 background markedly increased the survival time. Reciprocal congenic lines confirmed the opposing allelic effects of Shali1 and Shali2 on HALI survival time and provide unique models to identify their respective quantitative trait genes and to critically assess the apparent bidirectional epistatic interactions between these major-effect loci

Incompatibilities Involving Yeast Mismatch Repair Genes: A Role for Genetic Modifiers and Implications for Disease Penetrance and Variation in Genomic Mutation Rates

Genetic background effects underlie the penetrance of most genetically determined phenotypes, including human diseases. To explore how such effects can modify a mutant phenotype in a genetically tractable system, we examined an incompatibility involving the MLH1 and PMS1 mismatch repair genes using a large population sample of geographically and ecologically diverse Saccharomyces cerevisiae strains. The mismatch repair incompatibility segregates into naturally occurring yeast strains, with no strain bearing the deleterious combination. In assays measuring the mutator phenotype conferred by different combinations of MLH1 and PMS1 from these strains, we observed a mutator phenotype only in combinations predicted to be incompatible. Surprisingly, intragenic modifiers could be mapped that specifically altered the strength of the incompatibility over a 20-fold range. Together, these observations provide a powerful model in which to understand the basis of disease penetrance and how such genetic variation, created through mating, could result in new mutations that could be the raw material of adaptive evolution in yeast populations

Towards Response ADAptive Radiotherapy for organ preservation for intermediate-risk rectal cancer (preRADAR): protocol of a phase I dose-escalation trial

Introduction Organ preservation is associated with superior functional outcome and quality of life (QoL) compared with total mesorectal excision (TME) for rectal cancer. Only 10% of patients are eligible for organ preservation following short-course radiotherapy (SCRT, 25 Gy in five fractions) and a prolonged interval (4–8 weeks) to response evaluation. The organ preservation rate could potentially be increased by dose-escalated radiotherapy. Online adaptive magnetic resonance-guided radiotherapy (MRgRT) is anticipated to reduce radiation-induced toxicity and enable radiotherapy dose escalation. This trial aims to establish the maximum tolerated dose (MTD) of dose-escalated SCRT using online adaptive MRgRT.Methods and analysis The preRADAR is a multicentre phase I trial with a 6+3 dose-escalation design. Patients with intermediate-risk rectal cancer (cT3c-d(MRF-)N1M0 or cT1-3(MRF-)N1M0) interested in organ preservation are eligible. Patients are treated with a radiotherapy boost of 2×5 Gy (level 0), 3×5 Gy (level 1), 4×5 Gy (level 2) or 5×5 Gy (level 3) on the gross tumour volume in the week following standard SCRT using online adaptive MRgRT. The trial starts on dose level 1. The primary endpoint is the MTD based on the incidence of dose-limiting toxicity (DLT) per dose level. DLT is a composite of maximum one in nine severe radiation-induced toxicities and maximum one in three severe postoperative complications, in patients treated with TME or local excision within 26 weeks following start of treatment. Secondary endpoints include the organ preservation rate, non-DLT, oncological outcomes, patient-reported QoL and functional outcomes up to 2 years following start of treatment. Imaging and laboratory biomarkers are explored for early response prediction.Ethics and dissemination The trial protocol has been approved by the Medical Ethics Committee of the University Medical Centre Utrecht. The primary and secondary trial results will be published in international peer-reviewed journals.Biological, physical and clinical aspects of cancer treatment with ionising radiatio