Somatic mutations reveal asymmetric cellular dynamics in the early human embryo.

Somatic cells acquire mutations throughout the course of an individual's life. Mutations occurring early in embryogenesis are often present in a substantial proportion of, but not all, cells in postnatal humans and thus have particular characteristics and effects. Depending on their location in the genome and the proportion of cells they are present in, these mosaic mutations can cause a wide range of genetic disease syndromes and predispose carriers to cancer. They have a high chance of being transmitted to offspring as de novo germline mutations and, in principle, can provide insights into early human embryonic cell lineages and their contributions to adult tissues. Although it is known that gross chromosomal abnormalities are remarkably common in early human embryos, our understanding of early embryonic somatic mutations is very limited. Here we use whole-genome sequences of normal blood from 241 adults to identify 163 early embryonic mutations. We estimate that approximately three base substitution mutations occur per cell per cell-doubling event in early human embryogenesis and these are mainly attributable to two known mutational signatures. We used the mutations to reconstruct developmental lineages of adult cells and demonstrate that the two daughter cells of many early embryonic cell-doubling events contribute asymmetrically to adult blood at an approximately 2:1 ratio. This study therefore provides insights into the mutation rates, mutational processes and developmental outcomes of cell dynamics that operate during early human embryogenesis.

The circular RNome of primary breast cancer.

Circular RNAs (circRNAs) are a class of RNAs that is under increasing scrutiny, although their functional roles are debated. We analyzed RNA-seq data of 348 primary breast cancers and developed a method to identify circRNAs that does not rely on unmapped reads or known splice junctions. We identified 95,843 circRNAs, of which 20,441 were found recurrently. Of the circRNAs that match exon boundaries of the same gene, 668 showed a poor or even negative (R < 0.2) correlation with the expression level of the linear gene. In silico analysis showed only a minority (8.5%) of circRNAs could be explained by known splicing events. Both these observations suggest that specific regulatory processes for circRNAs exist. We confirmed the presence of circRNAs of CNOT2, CREBBP, and RERE in an independent pool of primary breast cancers. We identified circRNA profiles associated with subgroups of breast cancers and with biological and clinical features, such as amount of tumor lymphocytic infiltrate and proliferation index. siRNA-mediated knockdown of circCNOT2 was shown to significantly reduce viability of the breast cancer cell lines MCF-7 and BT-474, further underlining the biological relevance of circRNAs. Furthermore, we found that circular, and not linear, CNOT2 levels are predictive for progression-free survival time to aromatase inhibitor (AI) therapy in advanced breast cancer patients, and found that circCNOT2 is detectable in cell-free RNA from plasma. We showed that circRNAs are abundantly present, show characteristics of being specifically regulated, are associated with clinical and biological properties, and thus are relevant in breast cancer.

Impact of classical and basal-like molecular subtypes on overall survival in resected pancreatic cancer in the SPACIOUS-2 multicentre study.

BACKGROUND: The recently identified classical and basal-like molecular subtypes of pancreatic cancer impact on overall survival (OS). However, the added value of routine subtyping in both clinical practice and randomized trials is still unclear, as most studies do not consider clinicopathological parameters. This study examined the clinical prognostic value of molecular subtyping in patients with resected pancreatic cancer. METHODS: Subtypes were determined on fresh-frozen resected pancreatic cancer samples from three Dutch centres using the Purity Independent Subtyping of Tumours classification. Patient, treatment, and histopathological variables were compared between subtypes. The prognostic value of subtyping in (simulated) pre- and postoperative settings was assessed using Kaplan-Meier and Cox regression analyses. RESULTS: Of 199 patients with resected pancreatic cancer, 164 (82.4 per cent) were classified as the classical and 35 (17.6 per cent) as the basal-like subtype. Patients with a basal-like subtype had worse OS (11 versus 16 months (HR 1.49, 95 per cent c.i. 1.03 to 2.15; P = 0.035)) than patients with a classical subtype. In multivariable Cox regression analysis, including only clinical variables, the basal-like subtype was a statistically significant predictor for poor OS (HR 1.61, 95 per cent c.i. 1.11 to 2.34; P = 0.013). When histopathological variables were added to this model, the prognostic value of subtyping decreased (HR 1.49, 95 per cent c.i. 1.01 to 2.19; P = 0.045). CONCLUSION: The basal-like subtype was associated with worse OS in patients with resected pancreatic cancer. Adding molecular classification to inform on tumor biology may be used in patient stratification.

High-grade mesenchymal pancreatic ductal adenocarcinoma drives stromal deactivation through CSF-1.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by an abundance of stroma. Multiple molecular classification efforts have identified a mesenchymal tumor subtype that is consistently characterized by high-grade growth and poor clinical outcome. The relation between PDAC stroma and tumor subtypes is still unclear. Here, we aimed to identify how PDAC cells instruct the main cellular component of stroma, the pancreatic stellate cells (PSCs). We found in primary tissue that high-grade PDAC had reduced collagen deposition compared to low-grade PDAC. Xenografts and organotypic co-cultures established from mesenchymal-like PDAC cells featured reduced collagen and activated PSC content. Medium transfer experiments using a large set of PDAC cell lines revealed that mesenchymal-like PDAC cells consistently downregulated ACTA2 and COL1A1 expression in PSCs and reduced proliferation. We identified colony-stimulating factor 1 as the mesenchymal PDAC-derived ligand that deactivates PSCs, and inhibition of its receptor CSF1R was able to counteract this effect. In conclusion, high-grade PDAC features stroma that is low in collagen and activated PSC content, and targeting CSF1R offers direct options to maintain a tumor-restricting microenvironment.

Establishment of the Dutch Nationwide, Interdisciplinary Infrastructure and Biobank for Fundamental and Translational Ovarian Cancer Research: Archipelago of Ovarian Cancer Research.

OBJECTIVES: Ovarian cancer has the worst overall survival rate of all gynecologic malignancies. For the majority of patients, the 5-year overall survival rate of less than 50% has hardly improved over the last decades. To improve the outcome of patients with all subtypes of ovarian cancer, large-scale fundamental and translational research is needed. To accommodate these types of ovarian cancer research, we have established a Dutch nationwide, interdisciplinary infrastructure and biobank: the Archipelago of Ovarian Cancer Research (AOCR). The AOCR will facilitate fundamental and translational ovarian cancer research and enhance interdisciplinary, national, and international collaboration. DESIGN: The AOCR biobank is a prospective ovarian cancer biobank in which biomaterials are collected, processed, and stored in a uniform matter for future (genetic) scientific research. All 19 Dutch hospitals in which ovarian cancer surgery is performed participate and collaborate in the AOCR biobank. PARTICIPANTS/MATERIALS, SETTING, METHODS: Patients of 16 years and older with suspected or diagnosed ovarian, fallopian tube, or primary peritoneal cancer are recruited for participation. Patients who agree to participate give written informed consent for collection, storage, and issue of their biomaterials for future studies. After inclusion, different blood samples are taken at various predefined time points both before and during treatment. In case of a diagnostic paracentesis or biopsy, the residual biomaterials of these procedures are stored in the biobank. During surgery, primary tumor tissue and, if applicable, tissue from metastatic sites are collected and stored. From each patient, a representative histological hematoxylin and eosin stained slide is digitalized for research purposes, including reassessment by a panel of gynecologic pathologists. Clinical and pathological data are obtained on a per-study basis from Dutch registries. Research proposals for the issue of biomaterials and data are evaluated by both the Archipelago Scientific Committee and the Steering Committee. Researchers using the biomaterials from the AOCR biobank are encouraged to enrich the biobank with data and materials resulting from their analyses and experiments. LIMITATIONS: The implementation and first 4 years of collection are financed by an infrastructural grant from the Dutch Cancer Society. Therefore, the main limitation is that the costs for sustaining the biobank after the funding period will have to be covered. This coverage will come from incorporation of budget for biobanking in future grant applications and from fees from external researchers and commercial parties using the biomaterials stored in the AOCR biobank. Moreover, we will apply for grants aimed at sustaining and improving research infrastructures and biobanks. CONCLUSIONS: With the establishment of the Dutch nationwide, interdisciplinary Archipelago of Ovarian Cancer Research infrastructure and biobank, fundamental and translational research on ovarian cancer can be greatly improved. The ultimate aim of this infrastructure is that it will lead to improved diagnostics, treatment, and survival of patients with ovarian cancer.

Circulating tumor DNA quantity is related to tumor volume and both predict survival in metastatic pancreatic ductal adenocarcinoma.

Circulating tumor DNA (ctDNA) is assumed to reflect tumor burden and has been suggested as a tool for prognostication and follow-up in patients with metastatic pancreatic ductal adenocarcinoma (mPDAC). However, the prognostic value of ctDNA and its relation with tumor burden has yet to be substantiated, especially in mPDAC. In this retrospective analysis of prospectively collected samples, cell-free DNA from plasma samples of 58 treatment-naive mPDAC patients was isolated and sequenced using a custom-made pancreatobiliary NGS panel. Pathogenic mutations were detected in 26/58 (44.8%) samples. Cross-check with droplet digital PCR showed good agreement in Bland-Altman analysis (p = 0.217, nonsignificance indicating good agreement). In patients with liver metastases, ctDNA was more frequently detected (24/37, p < 0.001). Tumor volume (3D reconstructions from imaging) and ctDNA variant allele frequency (VAF) were correlated (Spearman's ρ = 0.544, p < 0.001). Median overall survival (OS) was 3.2 (95% confidence interval [CI] 1.6-4.9) versus 8.4 (95% CI 1.6-15.1) months in patients with detectable versus undetectable ctDNA (p = 0.005). Both ctDNA VAF and tumor volume independently predicted OS after adjustment for carbohydrate antigen 19.9 and treatment regimen (hazard ratio [HR] 1.05, 95% CI 1.01-1.09, p = 0.005; HR 1.00, 95% CI 1.01-1.05, p = 0.003). In conclusion, our study showed that ctDNA detection rates are higher in patients with larger tumor volume and liver metastases. Nevertheless, measurements may diverge and, thus, can provide complementary information. Both ctDNA VAF and tumor volume were strong predictors of OS.

Comprehensive evaluation of methods to assess overall and cell-specific immune infiltrates in breast cancer.

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.

Optoacoustic imaging of the breast: correlation with histopathology and histopathologic biomarkers.

AIM: This study was conducted in order to investigate the role of gray-scale ultrasound (US) and optoacoustic imaging combined with gray-scale ultrasound (OA/US) to better differentiate between breast cancer molecular subtypes. MATERIALS AND METHODS: All 67 malignant masses included in the Maestro trial were retrospectively reviewed to compare US and OA/US feature scores and histopathological findings. Kruskal-Wallis tests were used to analyze the relationship between US and OA/US features and molecular subtypes of breast cancer. If a significant relationship was found, additional Wilcoxon-Mann-Whitney tests were used to identify the differences between molecular subtype groups. RESULTS: US sound transmission helped to differentiate between LUMA and LUMB, LUMB and TNBC, and LUMB and all other molecular subtypes combined (p values < 0.05). Regarding OA/US features, the sum of internal features helped to differentiate between TNBC and HER2-enriched subtypes (p = 0.049). Internal vessels (p = 0.025), sum of all internal features (p = 0.019), and sum of internal and external features (p = 0.028) helped to differentiate between LUMA and LUMB. All internal features, the sum of all internal features, the sum of all internal and external features, and the ratio of internal and external features helped to differentiate between LUMA and TNBC. The same features also helped to differentiate between LUMA and TNBC from other molecular subtypes (p values < 0.05). CONCLUSIONS: The use of OA/US might help radiologists to better differentiate between breast cancer molecular subtypes. Further studies need to be carried out in order to validate these results. KEY POINTS: • The combination of functional and morphologic information provided by optoacoustic imaging (OA) combined with gray-scale US helped to differentiate between breast cancer molecular subtypes.

Downgrading of Breast Masses Suspicious for Cancer by Using Optoacoustic Breast Imaging.

Purpose To assess the ability of optoacoustic (OA) ultrasonography (US) to help correctly downgrade benign masses classified as Breast Imaging Reporting and Data System (BI-RADS) 4a and 4b to BI-RADS 3 or 2. Materials and Methods OA/US technology uses laser light to detect relative amounts of oxygenated and deoxygenated hemoglobin in and around suspicious breast masses. In this prospective, multicenter study, results of 209 patients with 215 breast masses classified as BI-RADS 4a or 4b at US are reported. Patients were enrolled between 2015 and 2016. Masses were first evaluated with US with knowledge of previous clinical information and imaging results, and from this information a US imaging-based probability of malignancy (POM) and BI-RADS category were assigned to each mass. The same masses were then re-evaluated at OA/US. During the OA/US evaluation, radiologists scored five OA/US features, and then reassigned an OA/US-based POM and BI-RADS category for each mass. BI-RADS downgrade and upgrade percentages at OA/US were assessed by using a weighted sum of the five OA feature scores. Results At OA/US, 47.9% (57 of 119; 95% CI: 0.39, 0.57) of benign masses classified as BI-RADS 4a and 11.1% (three of 27; 95% CI: 0.03, 0.28) of masses classified as BI-RADS 4b were correctly downgraded to BI-RADS 3 or 2. Two of seven malignant masses classified as BI-RADS 4a at US were incorrectly downgraded, and one of 60 malignant masses classified as BI-RADS 4b at US was incorrectly downgraded for a total of 4.5% (three of 67; 95% CI: 0.01, 0.13) false-negative findings. Conclusion At OA/US, benign masses classified as BI-RADS 4a could be downgraded in BI-RADS category, which would potentially decrease biopsies negative for cancer and short-interval follow-up examinations, with the limitation that a few masses may be inappropriately downgraded.

The landscape of cancer genes and mutational processes in breast cancer.

All cancers carry somatic mutations in their genomes. A subset, known as driver mutations, confer clonal selective advantage on cancer cells and are causally implicated in oncogenesis, and the remainder are passenger mutations. The driver mutations and mutational processes operative in breast cancer have not yet been comprehensively explored. Here we examine the genomes of 100 tumours for somatic copy number changes and mutations in the coding exons of protein-coding genes. The number of somatic mutations varied markedly between individual tumours. We found strong correlations between mutation number, age at which cancer was diagnosed and cancer histological grade, and observed multiple mutational signatures, including one present in about ten per cent of tumours characterized by numerous mutations of cytosine at TpC dinucleotides. Driver mutations were identified in several new cancer genes including AKT2, ARID1B, CASP8, CDKN1B, MAP3K1, MAP3K13, NCOR1, SMARCD1 and TBX3. Among the 100 tumours, we found driver mutations in at least 40 cancer genes and 73 different combinations of mutated cancer genes. The results highlight the substantial genetic diversity underlying this common disease.

Association between gene expression profile of the primary tumor and chemotherapy response of metastatic breast cancer.

BACKGROUND: To better predict the likelihood of response to chemotherapy, we have conducted a study comparing the gene expression patterns of primary tumours with their corresponding response to systemic chemotherapy in the metastatic setting. METHODS: mRNA expression profiles of breast carcinomas of patients that later developed distant metastases were analyzed using supervised and non-supervised classification techniques to identify predictors of response to chemotherapy. The top differentially expressed genes between the responders and non-responders were identified and further explored. An independent dataset which was generated to predict response to neo-adjuvant CT was utilized for the purpose of validation. Response to chemotherapy was also correlated to the clinicopathologic characteristics, molecular subtypes, metastatic behavior and survival outcomes. RESULTS: Anthracycline containing regimens were the most common first line treatment (58.4%), followed by non-anthracycline/non-taxane containing (25.8%) and taxane containing (15.7%) regimens. Response was achieved in 41.6% of the patients to the first line CT and in 21.8% to second line CT. Response was not found to be significantly correlated to tumour type, grade, lymph node status, ER and PR status. Patients with HER2+ tumours showed better response to anthracycline containing therapy (p: 0.002). Response to first and second line chemotherapy did not differ among gene expression based molecular subtypes (p: 0.236 and p: 0.20). Using supervised classification, a 14 gene response classifier was identified. This 14-gene predictor could successfully predict the likelihood of better response to first and second line CT (p: <.0001 and p: 0.761, respectively) in the training set. However, the predictive value of this gene set in data of response to neoadjuvant chemotherapy could not be validated. CONCLUSIONS: To our knowledge, this is the first study revealing the relation between gene expression profiles of the primary tumours and their chemotherapy responsiveness in the metastatic setting. In contrast to the findings for neoadjuvant chemotherapy treatment, there was no association of molecular subtype with response to chemotherapy in the metastatic setting. Using supervised classification, we identified a classifier of chemotherapy response; however, we could not validate this classifier using neoadjuvant response data. TRIAL REGISTRATION: Non applicable. Subjects were retrospectively registered.

A novel gene expression signature for bone metastasis in breast carcinomas.

Metastatic cancer remains the leading cause of death for patients with breast cancer. To understand the mechanisms underlying the development of distant metastases to specific sites is therefore important and of potential clinical value. From 157 primary breast tumours of the patients with known metastatic disease, gene expression profiling data were generated and correlated to metastatic behaviour including site-specific metastasis, metastasis pattern and survival outcomes. We analysed gene expression signatures specifically associated with the development of bone metastases. As a validation cohort, we used a published dataset of 376 breast carcinomas for which gene expression data and site-specific metastasis information were available. 80.5 % of luminal-type tumours developed bone metastasis as opposed to 41.7 % of basal and 55.6 % of HER2-like tumours. A novel 15-gene signature identified 82.4 % of the tumours with bone metastasis, 85.2 % of the tumours which had bone metastasis as first site of metastasis and 100 % of the ones with bone metastasis only (p 9.99e-09), in the training set. In the independent dataset, 81.2 % of the positive tested tumours had known metastatic disease to the bone (p 4.28e-10). This 15-gene signature showed much better correlation with the development of bone metastases than previously identified signatures and was predictive in both ER-positive as well as in ER-negative tumours. Multivariate analyses revealed that together with the molecular subtype, our 15-gene expression signature was significantly correlated to bone metastasis status (p <0.001, 95 % CI 3.86-48.02 in the training set; p 0.001, 95 % CI 1.54-5.00 in the independent set). The 15 genes, APOPEC3B, ATL2, BBS1, C6orf61, C6orf167, MMS22L, KCNS1, MFAP3L, NIP7, NUP155, PALM2, PH-4, PGD5, SFT2D2 and STEAP3, encoded mainly membrane-bound molecules with molecular function of protein binding. The expression levels of the up-regulated genes (NAT1, BBS1 and PH-4) were also found to be correlated to epithelial to mesenchymal transition status of the tumour. We have identified a novel 15-gene expression signature associated with the development of bone metastases in breast cancer patients. This bone metastasis signature is the first to be identified using a supervised classification approach in a large series of patients and will help forward research in this area towards clinical applications.

Integrated molecular pathway analysis informs a synergistic combination therapy targeting PTEN/PI3K and EGFR pathways for basal-like breast cancer.

BACKGROUND: The basal-like breast cancer (BLBC) subtype is characterized by positive staining for basal mammary epithelial cytokeratin markers, lack of hormone receptor and HER2 expression, and poor prognosis with currently no approved molecularly-targeted therapies. The oncogenic signaling pathways driving basal-like tumorigenesis are not fully elucidated. METHODS: One hundred sixteen unselected breast tumors were subjected to integrated analysis of phosphoinositide 3-kinase (PI3K) pathway related molecular aberrations by immunohistochemistry, mutation analysis, and gene expression profiling. Incidence and relationships between molecular biomarkers were characterized. Findings for select biomarkers were validated in an independent series. Synergistic cell killing in vitro and in vivo tumor therapy was investigated in breast cancer cell lines and mouse xenograft models, respectively. RESULTS: Sixty-four % of cases had an oncogenic alteration to PIK3CA, PTEN, or INPP4B; when including upstream kinases HER2 and EGFR, 75 % of cases had one or more aberration including 97 % of estrogen receptor (ER)-negative tumors. PTEN-loss was significantly associated to stathmin and EGFR overexpression, positivity for the BLBC markers cytokeratin 5/14, and the BLBC molecular subtype by gene expression profiling, informing a potential therapeutic combination targeting these pathways in BLBC. Combination treatment of BLBC cell lines with the EGFR-inhibitor gefitinib plus the PI3K pathway inhibitor LY294002 was synergistic, and correspondingly, in an in vivo BLBC xenograft mouse model, gefitinib plus PI3K-inhibitor PWT-458 was more effective than either monotherapy and caused tumor regression. CONCLUSIONS: Our study emphasizes the importance of PI3K/PTEN pathway activity in ER-negative and basal-like breast cancer and supports the future clinical evaluation of combining EGFR and PI3K pathway inhibitors for the treatment of BLBC.

Computer tomography colonography participation and yield in patients under surveillance for 6-9 mm polyps in a population-based screening trial.

PURPOSE: Surveillance CT colonography (CTC) is a viable option for 6-9 mm polyps at CTC screening for colorectal cancer. We established participation and diagnostic yield of surveillance and determined overall yield of CTC screening. MATERIAL AND METHODS: In an invitational CTC screening trial 82 of 982 participants harboured 6-9 mm polyps as the largest lesion(s) for which surveillance CTC was advised. Only participants with one or more lesion(s) ≥6 mm at surveillance CTC were offered colonoscopy (OC); 13 had undergone preliminary OC. The surveillance CTC yield was defined as the number of participants with advanced neoplasia in the 82 surveillance participants, and was added to the primary screening yield. RESULTS: Sixty-five of 82 participants were eligible for surveillance CTC of which 56 (86.2 %) participated. Advanced neoplasia was diagnosed in 15/56 participants (26.8 %) and 9/13 (69.2 %) with preliminary OC. Total surveillance yield was 24/82 (29.3 %). No carcinomas were detected. Adding surveillance results to initial screening CTC yield significantly increased the advanced neoplasia yield per 100 CTC participants (6.1 to 8.6; p < 0.001) and per 100 invitees (2.1 to 2.9; p < 0.001). CONCLUSION: Surveillance CTC for 6-9 mm polyps has a substantial yield of advanced adenomas and significantly increased the CTC yield in population screening. KEY POINTS: • The participation rate in surveillance CT colonography (CTC) is 86 %. • Advanced adenoma prevalence in a 6-9 mm CTC surveillance population is high. • Surveillance CTC significantly increases the yield of population screening by CTC. • Surveillance CTC for 6-9 mm polyps is a safe strategy. • Surveillance CTC is unlikely to yield new important extracolonic findings.

Long non-coding RNA HOTAIR reprograms chromatin state to promote cancer metastasis.

Large intervening non-coding RNAs (lincRNAs) are pervasively transcribed in the genome yet their potential involvement in human disease is not well understood. Recent studies of dosage compensation, imprinting, and homeotic gene expression suggest that individual lincRNAs can function as the interface between DNA and specific chromatin remodelling activities. Here we show that lincRNAs in the HOX loci become systematically dysregulated during breast cancer progression. The lincRNA termed HOTAIR is increased in expression in primary breast tumours and metastases, and HOTAIR expression level in primary tumours is a powerful predictor of eventual metastasis and death. Enforced expression of HOTAIR in epithelial cancer cells induced genome-wide re-targeting of Polycomb repressive complex 2 (PRC2) to an occupancy pattern more resembling embryonic fibroblasts, leading to altered histone H3 lysine 27 methylation, gene expression, and increased cancer invasiveness and metastasis in a manner dependent on PRC2. Conversely, loss of HOTAIR can inhibit cancer invasiveness, particularly in cells that possess excessive PRC2 activity. These findings indicate that lincRNAs have active roles in modulating the cancer epigenome and may be important targets for cancer diagnosis and therapy.

Evolution of Screen-Detected Small (6-9 mm) Polyps After a 3-Year Surveillance Interval: Assessment of Growth With CT Colonography Compared With Histopathology.

OBJECTIVES: Volumetric growth assessment has been proposed for predicting advanced histology at surveillance computed tomography (CT) colonography (CTC). We examined whether is it possible to predict which small (6-9 mm) polyps are likely to become advanced adenomas at surveillance by assessing volumetric growth. METHODS: In an invitational population-based CTC screening trial, 93 participants were diagnosed with one or two 6-9 mm polyps as the largest lesion(s). They were offered a 3-year surveillance CTC. Participants in whom surveillance CTC showed lesion(s) of ≥6 mm were offered colonoscopy. Volumetric measurements were performed on index and surveillance CTC, and polyps were classified into growth categories according to ±30% volumetric change (>30% growth as progression, 30% growth to 30% decrease as stable, and >30% decrease as regression). Polyp growth was related to histopathology. RESULTS: Between July 2012 and May 2014, 70 patients underwent surveillance CTC after a mean surveillance interval of 3.3 years (s.d. 0.3; range 3.0-4.6 years). In all, 33 (35%) of 95 polyps progressed, 36 (38%) remained stable, and 26 (27%) regressed, including an apparent resolution in 13 (14%) polyps. In 68 (83%) of the 82 polyps at surveillance, histopathology was obtained; 15 (47%) of 32 progressing polyps were advanced adenomas, 6 (21%) of 28 stable polyps, and none of the regressing polyps. CONCLUSIONS: The majority of 6-9 mm polyps will not progress to advanced neoplasia within 3 years. Those that do progress to advanced status can in particular be found among the lesions that increased in size on surveillance CTC.

Retrospective analysis of metastatic behaviour of breast cancer subtypes.

Among breast cancer patients who develop distant metastases, there is marked variability in the clinical course, including metastasis pattern. Here, we present a retrospective study of breast cancer patients who all developed distant metastases focusing on the association between breast cancer subtype and clinical course, including organ-specific metastasis. Tissue microarrays (TMAs) were assembled and stained for ER, PR, HER2, EGFR, CK5/6, CK14, E-Cadherin, TP53 and Ki67 for 263 breast cancer patients with metastatic disease. Tumours were classified into ER+/HER2-/Ki67high, ER+/HER2-/Ki67low, ER+/HER2+, ER-/HER2+ and ER-/HER2- groups. Relevant data related to metastasis pattern, metastasis timeline, systemic treatment and survival were retrieved. Associations between site-specific relapse and patient/tumour characteristics were assessed with multivariate models using logistic regression. Median time for development of distant metastasis was 30 months (range 0-15.3 years); 75.8 % of the distance metastases developed in the first 5 years after treatment of the primary tumour. Patients with ER-/HER2- tumours had a median overall survival of 27 months; those with HER2+ tumours of 52 months; those with ER+/HER2-/Ki67high of 76 months and those with ER+/HER2-/Ki67low of 79 months. Bone was the most common site for distant metastasis (70.6 %) followed by liver (54.5 %) and lung (31.4 %), respectively. Visceral metastasis was found in 76.8 % of the patients. Patients with ER-/HER2- tumours developed visceral metastases in 81 % and bone metastases in 55.2 %; those with HER2+ tumours developed visceral metastases in 77.4 % and bone metastases in 69.8 %; those with ER+/HER2-/Ki67high developed visceral metastases in 75.7 % and bone metastases in 87.8 % and those with ER+/HER2-/Ki67low developed visceral metastases in 76.9 % and bone metastases in 73.1 %. In metastatic breast cancer patients, tumour subtypes are associated with survival and pattern of distant metastases. These associations are of help in choices for surveillance and therapy in individual patients.

Establishment of patient-derived xenograft models and cell lines for malignancies of the upper gastrointestinal tract.

BACKGROUND: The upper gastrointestinal tract is home to some of most notorious cancers like esophagogastric and pancreatic cancer. Several factors contribute to the lethality of these tumors, but one that stands out for both tumor types is the strong inter- as well as intratumor heterogeneity. Unfortunately, genetic tumor models do not match this heterogeneity, and for esophageal cancer no adequate genetic models exist. To allow for an improved understanding of these diseases, tissue banks with sufficient amount of samples to cover the extent of diversity of human cancers are required. Additionally, xenograft models that faithfully mimic and span the breadth of human disease are essential to perform meaningful functional experiments. METHODS: We describe here the establishment of a tissue biobank, patient derived xenografts (PDXs) and cell line models of esophagogastric and pancreatic cancer patients. Biopsy material was grafted into immunocompromised mice and PDXs were used to establish primary cell cultures to perform functional studies. Expression of Hedgehog ligands in patient tumor and matching PDX was assessed by immunohistochemical staining, and quantitative real-time PCR as well as flow cytometry was used for cultured cells. Cocultures with Hedgehog reporter cells were performed to study paracrine signaling potency. Furthermore, SHH expression was modulated in primary cultures using lentiviral mediated knockdown. RESULTS: We have established a panel of 29 PDXs from esophagogastric and pancreatic cancers, and demonstrate that these PDXs mirror several of the (immuno)histological and biochemical characteristics of the original tumors. Derived cell lines can be genetically manipulated and used to further study tumor biology and signaling capacity. In addition, we demonstrate an active (paracrine) Hedgehog signaling mode by both tumor types, the magnitude of which has not been compared directly in previous studies. CONCLUSIONS: Our established PDXs and their matching primary cell lines retain important characteristics seen in the original tumors, and this should enable future studies to address the responses of these tumors to different treatment modalities, but also help in gaining mechanistic insight in how some tumors respond to certain regimens and others do not.

Papillary and neuroendocrine breast lesions: the WHO stance.

In this review, we highlight adaptations in the WHO 2012 classification of papillary and neuroendocrine breast lesions as compared with the previous 2003 version. Consensus criteria for distinguishing atypical ductal hyperplasia from ductal carcinoma in situ within an intraductal papilloma are proposed. The absence of myoepithelial cells around the wall of an encapsulated papillary carcinoma, although raising consideration of an indolent tumour with minimal invasion, is currently regarded as in-situ disease for staging purposes. The majority of solid papillary carcinomas are classified as in-situ tumours, but lesions with irregular tumour islands within desmoplastic stroma may be considered to be invasive. The diagnosis of solid papillary carcinoma without further qualification as either in-situ or invasive disease is discouraged. When invasive papillary carcinoma is seen in the breast, metastatic papillary carcinoma from other organ sites needs to be excluded. WHO 2012 classifies neuroendocrine breast tumours as well-differentiated neuroendocrine tumour, small-cell carcinoma, and invasive breast carcinoma with neuroendocrine differentiation. There is currently no clinical impact of identifying neuroendocrine differentiation in conventional invasive breast carcinomas, apart from acknowledging its frequent occurrence in subtypes such as the hypercellular variant of mucinous carcinoma and solid papillary carcinoma.