Publisher Correction: MethCORR modelling of methylomes from formalin-fixed paraffin-embedded tissue enables characterization and prognostication of colorectal cancer.

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MethCORR modelling of methylomes from formalin-fixed paraffin-embedded tissue enables characterization and prognostication of colorectal cancer.

Transcriptional characterization and classification has potential to resolve the inter-tumor heterogeneity of colorectal cancer and improve patient management. Yet, robust transcriptional profiling is difficult using formalin-fixed, paraffin-embedded (FFPE) samples, which complicates testing in clinical and archival material. We present MethCORR, an approach that allows uniform molecular characterization and classification of fresh-frozen and FFPE samples. MethCORR identifies genome-wide correlations between RNA expression and DNA methylation in fresh-frozen samples. This information is used to infer gene expression information in FFPE samples from their methylation profiles. MethCORR is here applied to methylation profiles from 877 fresh-frozen/FFPE samples and comparative analysis identifies the same two subtypes in four independent cohorts. Furthermore, subtype-specific prognostic biomarkers that better predicts relapse-free survival (HR = 2.66, 95%CI [1.67-4.22], P value < 0.001 (log-rank test)) than UICC tumor, node, metastasis (TNM) staging and microsatellite instability status are identified and validated using DNA methylation-specific PCR. The MethCORR approach is general, and may be similarly successful for other cancer types.

Characterization of genetic intratumor heterogeneity in colorectal cancer and matching patient-derived spheroid cultures.

Patient-derived in vitro cultures of colorectal cancer (CRC) may help guide treatment strategies prior to patient treatment. However, most previous studies have been performed on a single biopsy per tumor. The purpose of this study was to analyze multiple spatially distinct biopsies from CRCs and see how well intratumor heterogeneity (ITH) was recapitulated in matching patient-derived spheroids. Three to five biopsies were collected from six CRC tumors. Each biopsy was split in two; one half was used for spheroid culturing, while the other half was used for DNA and RNA purification. For two patients, lymph node metastases were analyzed. Somatic mutations were called from whole exome sequencing data. Each tumor contained mutations shared across all biopsies and spheroids, including major CRC drivers such as APC, KRAS, and TP53. At the same time, all tumors exhibited ITH on both mutation and copy number level. The concordance between biopsies and spheroids ranged between 40 and 70% for coding mutations. For three patients, the biopsy and spheroid from matching areas clustered together, meaning that the spheroid resembled the area of origin more than the other areas. However, all biopsies and spheroids contained private mutations. Therefore, multiple cultures from spatially distinct sites of the tumor increase the insight into the genetic profile of the entire tumor. Molecular subtypes were called from RNA sequencing data. When based on transcripts from both cancer and noncancerous cells, the subtypes were largely independent of sampling site. In contrast, subtyping based on cancer cell transcripts alone was dependent on sample site and genetic ITH. In conclusion, all examined CRC tumors showed genetic ITH. Spheroid cultures partly reflected this ITH, and having multiple cultures from distinct tumor sites improved the representation of the genetic tumor subclones. This should be taken into account when establishing patient-derived models for drug screening.

A genetically inducible porcine model of intestinal cancer.

Transgenic porcine cancer models bring novel possibilities for research. Their physical similarities with humans enable the use of surgical procedures and treatment approaches used for patients, which facilitates clinical translation. Here, we aimed to develop an inducible oncopig model of intestinal cancer. Transgenic (TG) minipigs were generated using somatic cell nuclear transfer by handmade cloning. The pigs encode two TG cassettes: (a) an Flp recombinase-inducible oncogene cassette containing KRAS-G12D, cMYC, SV40LT - which inhibits p53 - and pRB and (b) a 4-hydroxytamoxifen (4-OHT)-inducible Flp recombinase activator cassette controlled by the intestinal epithelium-specific villin promoter. Thirteen viable transgenic minipigs were born. The ability of 4-OHT to activate the oncogene cassette was confirmed in vitro in TG colonic organoids and ex vivo in tissue biopsies obtained by colonoscopy. In order to provide proof of principle that the oncogene cassette could also successfully be activated in vivo, three pigs were perorally treated with 400 mg tamoxifen for 2 × 5 days. After two months, one pig developed a duodenal neuroendocrine carcinoma with a lymph node metastasis. Molecular analysis of the carcinoma and metastasis confirmed activation of the oncogene cassette. No tumor formation was observed in untreated TG pigs or in the remaining two treated pigs. The latter indicates that tamoxifen delivery can probably be improved. In summary, we have generated a novel inducible oncopig model of intestinal cancer, which has the ability to form metastatic disease already two months after induction. The model may be helpful in bridging the gap between basic research and clinical usage. It opens new venues for longitudinal studies of tumor development and evolution, for preclinical assessment of new anticancer regimens, for pharmacology and toxicology assessments, as well as for studies into biological mechanisms of tumor formation and metastasis.

Clinical Implications of Monitoring Circulating Tumor DNA in Patients with Colorectal Cancer.

Purpose: We investigated whether detection of ctDNA after resection of colorectal cancer identifies the patients with the highest risk of relapse and, furthermore, whether longitudinal ctDNA analysis allows early detection of relapse and informs about response to intervention.Experimental Design: In this longitudinal cohort study, we used massively parallel sequencing to identify somatic mutations and used these as ctDNA markers to detect minimal residual disease and to monitor changes in tumor burden during a 3-year follow-up period.Results: A total of 45 patients and 371 plasma samples were included. Longitudinal samples from 27 patients revealed ctDNA postoperatively in all relapsing patients (n = 14), but not in any of the nonrelapsing patients. ctDNA detected relapse with an average lead time of 9.4 months compared with CT imaging. Of 21 patients treated for localized disease, six had ctDNA detected within 3 months after surgery. All six later relapsed compared with four of the remaining patients [HR, 37.7; 95% confidence interval (CI), 4.2-335.5; P < 0.001]. The ability of a 3-month ctDNA analysis to predict relapse was confirmed in 23 liver metastasis patients (HR 4.9; 95% CI, 1.5-15.7; P = 0.007). Changes in ctDNA levels induced by relapse intervention (n = 19) showed good agreement with changes in tumor volume (κ = 0.41; Spearman ρ = 0.4).Conclusions: Postoperative ctDNA detection provides evidence of residual disease and identifies patients at very high risk of relapse. Longitudinal surveillance enables early detection of relapse and informs about response to intervention. These observations have implications for the postoperative management of colorectal cancer patients. Clin Cancer Res; 23(18); 5437-45. ©2017 AACR.

Renal epithelial cells can release ATP by vesicular fusion.

Renal epithelial cells have the ability to release nucleotides as paracrine factors. In the intercalated cells of the collecting duct, ATP is released by connexin30 (cx30), which is selectively expressed in this cell type. However, ATP is released by virtually all renal epithelia and the aim of the present study was to identify possible alternative nucleotide release pathways in a renal epithelial cell model. We used MDCK (type1) cells to screen for various potential ATP release pathways. In these cells, inhibition of the vesicular H(+)-ATPases (bafilomycin) reduced both the spontaneous and hypotonically (80%)-induced nucleotide release. Interference with vesicular fusion using N-ethylamide markedly reduced the spontaneous nucleotide release, as did interference with trafficking from the endoplasmic reticulum to the Golgi apparatus (brefeldin A1) and vesicular transport (nocodazole). These findings were substantiated using a siRNA directed against SNAP-23, which significantly reduced spontaneous ATP release. Inhibition of pannexin and connexins did not affect the spontaneous ATP release in this cell type, which consists of ~90% principal cells. TIRF-microscopy of either fluorescently-labeled ATP (MANT-ATP) or quinacrine-loaded vesicles, revealed that spontaneous release of single vesicles could be promoted by either hypoosmolality (50%) or ionomycin. This vesicular release decreased the overall cellular fluorescence by 5.8 and 7.6% respectively. In summary, this study supports the notion that spontaneous and induced ATP release can occur via exocytosis in renal epithelial cells.

Agonists that increase [Ca²âº](i) halt the movement of acidic cytoplasmic vesicles in MDCK cells.

Translocation of vesicles within the cytoplasm is essential to normal cell function. The vesicles are typically transported along the microtubules to their destination. The aim of this study was to characterize the vesicular movement in resting and stimulated renal epithelial cells. MDCK cells loaded with either quinacrine or acridine orange, dyes taken up by acidic vesicles, were observed at 37°C in semiopen perfusion chambers. Time-lapse series were analyzed by Imaris software. Our data revealed vigorous movement of stained vesicles in resting MDCK cells. These movements seem to require intact microtubules because nocodazole leads to a considerable reduction of the vesicular movements. Interestingly, we found that extracellular ATP caused the vesicular movement to cease. This observation was obvious in time lapse. Similarly, other stimuli known to increase the intracellular Ca²âº concentration ([Ca²âº](i)) in MDCK cells (increment in the fluid flow rate or arginine vasopressin) also reduced the vesicular movement. These findings were quantified by analysis of single vesicular movement patterns. In this way, ATP was found to reduce the lateral displacement of the total population of vesicles by 40%. Because all these perturbations increase [Ca²âº](i), we speculated that this increase in [Ca²âº](i) was responsible for the vesicle arrest. Therefore, we tested the effect of the Ca²âº ionophore, ionomycin (1 µM), which in the presence of extracellular Ca²âº resulted in a considerable and sustained reduction of vesicular movement amounting to a 58% decrease in average lateral vesicular displacement. Our data suggest that vesicles transported on microtubules are paused when subjected to high intracellular Ca²âº concentrations. This may provide an additional explanation for the cytotoxic effect of high [Ca²âº](i).