Impact of molecular profile on prognosis and relapse pattern in low and intermediate risk endometrial cancer.

INTRODUCTION: The role of molecular classification in patients with low/intermediate risk endometrial cancer (EC) is uncertain. Higher precision in diagnostics will inform the unsettled debate on optimal adjuvant treatment. We aimed to determine the association of molecular profiling with patterns of relapse and survival. MATERIAL AND METHODS: This retrospective cohort study included patients referred to The Norwegian Radium Hospital, Oslo University Hospital from 2006-2017. Patients with low/intermediate risk EC were molecularly classified as pathogenic polymerase epsilon (POLE)-mutated, mismatch repair deficient (MMRd), p53 abnormal, or no specific molecular profile (NSMP). The main outcomes were time to recurrence (TTR) and cancer-specific survival (CSS). RESULTS: Of 626 patients, 610 could be molecularly classified. Fifty-seven patients (9%) had POLE-mutated tumors, 202 (33%) had MMRd tumors, 34 (6%) had p53 abnormal tumors and 317 (52%) had NSMP tumors. After median follow-up time of 8.9 years, there was a statistically significant difference in TTR and CSS by molecular groups. Patients with p53 abnormal tumors had poor prognosis, with 10 of the 12 patients with relapse presenting with para-aortic/distant metastases. Patients with POLE mutations had excellent prognosis. In the NSMP group, L1CAM expression was associated with shorter CSS but not TTR. CONCLUSIONS: The differences in outcome by molecular groups are driven by differences in relapse frequency and -patterns and demand a higher precision in diagnostics, also in patients with low/intermediate risk EC. Tailored adjuvant treatment strategies need to consider systemic treatment for patients with p53 abnormal tumors and de-escalated treatment for patients with POLE mutated tumors.

A clinical decision support system optimising adjuvant chemotherapy for colorectal cancers by integrating deep learning and pathological staging markers: a development and validation study.

BACKGROUND: The DoMore-v1-CRC marker was recently developed using deep learning and conventional haematoxylin and eosin-stained tissue sections, and was observed to outperform established molecular and morphological markers of patient outcome after primary colorectal cancer resection. The aim of the present study was to develop a clinical decision support system based on DoMore-v1-CRC and pathological staging markers to facilitate individualised selection of adjuvant treatment. METHODS: We estimated cancer-specific survival in subgroups formed by pathological tumour stage (pT<4 or pT4), pathological nodal stage (pN0, pN1, or pN2), number of lymph nodes sampled (≤12 or >12) if not pN2, and DoMore-v1-CRC classification (good, uncertain, or poor prognosis) in 997 patients with stage II or III colorectal cancer considered to have no residual tumour (R0) from two community-based cohorts in Norway and the UK, and used these data to define three risk groups. An external cohort of 1075 patients with stage II or III R0 colorectal cancer from the QUASAR 2 trial was used for validation; these patients were treated with single-agent capecitabine. The proposed risk stratification system was evaluated using Cox regression analysis. We similarly evaluated a risk stratification system intended to reflect current guidelines and clinical practice. The primary outcome was cancer-specific survival. FINDINGS: The new risk stratification system provided a hazard ratio of 10·71 (95% CI 6·39-17·93; p<0·0001) for high-risk versus low-risk patients and 3·06 (1·73-5·42; p=0·0001) for intermediate versus low risk in the primary analysis of the validation cohort. Estimated 3-year cancer-specific survival was 97·2% (95% CI 95·1-98·4; n=445 [41%]) for the low-risk group, 94·8% (91·7-96·7; n=339 [32%]) for the intermediate-risk group, and 77·6% (72·1-82·1; n=291 [27%]) for the high-risk group. The guideline-based risk grouping was observed to be less prognostic and informative (the low-risk group comprised only 142 [13%] of the 1075 patients). INTERPRETATION: Integrating DoMore-v1-CRC and pathological staging markers provided a clinical decision support system that risk stratifies more accurately than its constituent elements, and identifies substantially more patients with stage II and III colorectal cancer with similarly good prognosis as the low-risk group in current guidelines. Avoiding adjuvant chemotherapy in these patients might be safe, and could reduce morbidity, mortality, and treatment costs. FUNDING: The Research Council of Norway.

PTEN and DNA Ploidy Status by Machine Learning in Prostate Cancer.

Machine learning (ML) is expected to improve biomarker assessment. Using convolution neural networks, we developed a fully-automated method for assessing PTEN protein status in immunohistochemically-stained slides using a radical prostatectomy (RP) cohort (n = 253). It was validated according to a predefined protocol in an independent RP cohort (n = 259), alone and by measuring its prognostic value in combination with DNA ploidy status determined by ML-based image cytometry. In the primary analysis, automatically assessed dichotomized PTEN status was associated with time to biochemical recurrence (TTBCR) (hazard ratio (HR) = 3.32, 95% CI 2.05 to 5.38). Patients with both non-diploid tumors and PTEN-low had an HR of 4.63 (95% CI 2.50 to 8.57), while patients with one of these characteristics had an HR of 1.94 (95% CI 1.15 to 3.30), compared to patients with diploid tumors and PTEN-high, in univariable analysis of TTBCR in the validation cohort. Automatic PTEN scoring was strongly predictive of the PTEN status assessed by human experts (area under the curve 0.987 (95% CI 0.968 to 0.994)). This suggests that PTEN status can be accurately assessed using ML, and that the combined marker of automatically assessed PTEN and DNA ploidy status may provide an objective supplement to the existing risk stratification factors in prostate cancer.

Stromal composition predicts recurrence of early rectal cancer after local excision.

AIMS: After local excision of early rectal cancer, definitive lymph node status is not available. An alternative means for accurate assessment of recurrence risk is required to determine the most appropriate subsequent management. Currently used measures are suboptimal. We assess three measures of tumour stromal content to determine their predictive value after local excision in a well-characterised cohort of rectal cancer patients without prior radiotherapy. METHODS AND RESULTS: A total of 143 patients were included. Haematoxylin and eosin (H&E) sections were scanned for (i) deep neural network (DNN, a machine-learning algorithm) tumour segmentation into compartments including desmoplastic stroma and inflamed stroma; and (ii) digital assessment of tumour stromal fraction (TSR) and optical DNA ploidy analysis. 3' mRNA sequencing was performed to obtain gene expression data from which stromal and immune scores were calculated using the ESTIMATE method. Full results were available for 139 samples and compared with disease-free survival. All three methods were prognostic. Most strongly predictive was a DNN-determined ratio of desmoplastic to inflamed stroma >5.41 (P < 0.0001). A ratio of ESTIMATE stromal to immune score <1.19 was also predictive of disease-free survival (P = 0.00051), as was stromal fraction >36.5% (P = 0.037). CONCLUSIONS: The DNN-determined ratio of desmoplastic to inflamed ratio is a novel and powerful predictor of disease recurrence in locally excised early rectal cancer. It can be assessed on a single H&E section, so could be applied in routine clinical practice to improve the prognostic information available to patients and clinicians to inform the decision concerning further management.

Deep learning for prediction of colorectal cancer outcome: a discovery and validation study.

BACKGROUND: Improved markers of prognosis are needed to stratify patients with early-stage colorectal cancer to refine selection of adjuvant therapy. The aim of the present study was to develop a biomarker of patient outcome after primary colorectal cancer resection by directly analysing scanned conventional haematoxylin and eosin stained sections using deep learning. METHODS: More than 12 000 000 image tiles from patients with a distinctly good or poor disease outcome from four cohorts were used to train a total of ten convolutional neural networks, purpose-built for classifying supersized heterogeneous images. A prognostic biomarker integrating the ten networks was determined using patients with a non-distinct outcome. The marker was tested on 920 patients with slides prepared in the UK, and then independently validated according to a predefined protocol in 1122 patients treated with single-agent capecitabine using slides prepared in Norway. All cohorts included only patients with resectable tumours, and a formalin-fixed, paraffin-embedded tumour tissue block available for analysis. The primary outcome was cancer-specific survival. FINDINGS: 828 patients from four cohorts had a distinct outcome and were used as a training cohort to obtain clear ground truth. 1645 patients had a non-distinct outcome and were used for tuning. The biomarker provided a hazard ratio for poor versus good prognosis of 3·84 (95% CI 2·72-5·43; p<0·0001) in the primary analysis of the validation cohort, and 3·04 (2·07-4·47; p<0·0001) after adjusting for established prognostic markers significant in univariable analyses of the same cohort, which were pN stage, pT stage, lymphatic invasion, and venous vascular invasion. INTERPRETATION: A clinically useful prognostic marker was developed using deep learning allied to digital scanning of conventional haematoxylin and eosin stained tumour tissue sections. The assay has been extensively evaluated in large, independent patient populations, correlates with and outperforms established molecular and morphological prognostic markers, and gives consistent results across tumour and nodal stage. The biomarker stratified stage II and III patients into sufficiently distinct prognostic groups that potentially could be used to guide selection of adjuvant treatment by avoiding therapy in very low risk groups and identifying patients who would benefit from more intensive treatment regimes. FUNDING: The Research Council of Norway.

Prognostic value of mitotic checkpoint protein BUB3, cyclin B1, and pituitary tumor-transforming 1 expression in prostate cancer.

The mitotic checkpoint protein BUB3, cyclin B1 (CCNB1) and pituitary tumor-transforming 1 (PTTG1) regulates cell division, and are sparsely studied in prostate cancer. Deregulation of these genes can lead to genomic instability, a characteristic of more aggressive tumors. We aimed to determine the expression levels of BUB3, CCNB1, and PTTG1 as potential prognostic markers of recurrence after radical prostatectomy. Protein levels were determined by immunohistochemistry on three formalin-fixed paraffin-embedded tissue sections from each of the 253 patients treated with radical prostatectomy. Immunohistochemistry scores were obtained by automated image analysis for CCNB1 and PTTG1. Recurrence, defined as locoregional recurrence, distant metastasis or death from prostate cancer, was used as endpoint for survival analysis. Tumors having both positive and negative tumor areas for cytoplasmic BUB3 (30%), CCNB1 (28%), or PTTG1 (35%) were considered heterogeneous. Patients with ≥1 positive tumor area had significantly increased risk of disease recurrence in univariable analysis compared with patients where all tumor areas were negative for cytoplasmic BUB3 (hazard ratio [HR] = 2.18, 95% confidence interval [CI] 1.41-3.36), CCNB1 (HR = 2.98, 95% CI 1.93-4.61) and PTTG1 (HR = 1.91, 95% CI 1.23-2.97). Combining the scores of cytoplasmic BUB3 and CCNB1 improved risk stratification when integrated with the Cancer of the Prostate Risk Assessment post-Surgical (CAPRA-S) score (difference in concordance index = 0.024, 95% CI 0.001-0.05). In analysis of multiple tumor areas, prognostic value was observed for cytoplasmic BUB3, CCNB1, and PTTG1.

Chromatin organisation and cancer prognosis: a pan-cancer study.

BACKGROUND: Chromatin organisation affects gene expression and regional mutation frequencies and contributes to carcinogenesis. Aberrant organisation of DNA has been correlated with cancer prognosis in analyses of the chromatin component of tumour cell nuclei using image texture analysis. As yet, the methodology has not been sufficiently validated to permit its clinical application. We aimed to define and validate a novel prognostic biomarker for the automatic detection of heterogeneous chromatin organisation. METHODS: Machine learning algorithms analysed the chromatin organisation in 461 000 images of tumour cell nuclei stained for DNA from 390 patients (discovery cohort) treated for stage I or II colorectal cancer at the Aker University Hospital (Oslo, Norway). The resulting marker of chromatin heterogeneity, termed Nucleotyping, was subsequently independently validated in six patient cohorts: 442 patients with stage I or II colorectal cancer in the Gloucester Colorectal Cancer Study (UK); 391 patients with stage II colorectal cancer in the QUASAR 2 trial; 246 patients with stage I ovarian carcinoma; 354 patients with uterine sarcoma; 307 patients with prostate carcinoma; and 791 patients with endometrial carcinoma. The primary outcome was cancer-specific survival. FINDINGS: In all patient cohorts, patients with chromatin heterogeneous tumours had worse cancer-specific survival than patients with chromatin homogeneous tumours (univariable analysis hazard ratio [HR] 1·7, 95% CI 1·2-2·5, in the discovery cohort; 1·8, 1·0-3·0, in the Gloucester validation cohort; 2·2, 1·1-4·5, in the QUASAR 2 validation cohort; 3·1, 1·9-5·0, in the ovarian carcinoma cohort; 2·5, 1·8-3·4, in the uterine sarcoma cohort; 2·3, 1·2-4·6, in the prostate carcinoma cohort; and 4·3, 2·8-6·8, in the endometrial carcinoma cohort). After adjusting for established prognostic patient characteristics in multivariable analyses, Nucleotyping was prognostic in all cohorts except for the prostate carcinoma cohort (HR 1·7, 95% CI 1·1-2·5, in the discovery cohort; 1·9, 1·1-3·2, in the Gloucester validation cohort; 2·6, 1·2-5·6, in the QUASAR 2 cohort; 1·8, 1·1-3·0, for ovarian carcinoma; 1·6, 1·0-2·4, for uterine sarcoma; 1·43, 0·68-2·99, for prostate carcinoma; and 1·9, 1·1-3·1, for endometrial carcinoma). Chromatin heterogeneity was a significant predictor of cancer-specific survival in microsatellite unstable (HR 2·9, 95% CI 1·0-8·4) and microsatellite stable (1·8, 1·2-2·7) stage II colorectal cancer, but microsatellite instability was not a significant predictor of outcome in chromatin homogeneous (1·3, 0·7-2·4) or chromatin heterogeneous (0·8, 0·3-2·0) stage II colorectal cancer. INTERPRETATION: The consistent prognostic prediction of Nucleotyping in different biological and technical circumstances suggests that the marker of chromatin heterogeneity can be reliably assessed in routine clinical practice and could be used to objectively assist decision making in a range of clinical settings. An immediate application would be to identify high-risk patients with stage II colorectal cancer who might have greater absolute benefit from adjuvant chemotherapy. Clinical trials are warranted to evaluate the survival benefit and cost-effectiveness of using Nucleotyping to guide treatment decisions in multiple clinical settings. FUNDING: The Research Council of Norway, the South-Eastern Norway Regional Health Authority, the National Institute for Health Research, and the Wellcome Trust.

Global gene expression analysis reveals a link between NDRG1 and vesicle transport.

N-myc downstream-regulated gene 1 (NDRG1) is induced by cellular stress such as hypoxia and DNA damage, and in humans, germ line mutations cause Charcot-Marie-Tooth disease. However, the cellular roles of NDRG1 are not fully understood. Previously, NDRG1 was shown to mediate doxorubicin resistance under hypoxia, suggesting a role for NDRG1 in cell survival under these conditions. We found decreased apoptosis in doxorubicin-treated cells expressing NDRG1 shRNAs under normoxia, demonstrating a requirement for NDRG1 in apoptosis in breast epithelial cells under normal oxygen pressure. Also, different cellular stress regimens, such as hypoxia and doxorubicin treatment, induced NDRG1 through different stress signalling pathways. We further compared expression profiles in human breast epithelial cells ectopically over-expressing NDRG1 with cells expressing NDRG1 shRNAs in order to identify biological pathways where NDRG1 is involved. The results suggest that NDRG1 may have roles connected to vesicle transport.

Live imaging of drug responses in the tumor microenvironment in mouse models of breast cancer.

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.

Imaging tumor-stroma interactions during chemotherapy reveals contributions of the microenvironment to resistance.

Little is known about the dynamics of cancer cell death in response to therapy in the tumor microenvironment. Intravital microscopy of chemotherapy-treated mouse mammary carcinomas allowed us to follow drug distribution, cell death, and tumor-stroma interactions. We observed associations between vascular leakage and response to doxorubicin, including improved response in matrix metalloproteinase-9 null mice that had increased vascular leakage. Furthermore, we observed CCR2-dependent infiltration of myeloid cells after treatment and that Ccr2 null host mice responded better to treatment with doxorubicin or cisplatin. These data show that the microenvironment contributes critically to drug response via regulation of vascular permeability and innate immune cell infiltration. Thus, live imaging can be used to gain insights into drug responses in situ.

Regulated expression of a transgene introduced on an oriP/EBNA-1 PAC shuttle vector into human cells.

BACKGROUND: Sequencing of the human genome has led to most genes being available in BAC or PAC vectors. However, limited functional information has been assigned to most of these genes. Techniques for the manipulation and transfer of complete functional units on large DNA fragments into human cells are crucial for the analysis of complete genes in their natural genomic context. One limitation of the functional studies using these vectors is the low transfection frequency. RESULTS: We have constructed a shuttle vector, pPAC7, which contains both the EBNA-1 gene and oriP from the Epstein-Barr virus allowing stable maintenance of PAC clones in the nucleus of human cells. The pPAC7 vector also contains the EGFP reporter gene, which allows direct monitoring of the presence of PAC constructs in transfected cells, and the Bsr-cassette that allows highly efficient and rapid selection in mammalian cells by use of blasticidin. Positive selection for recombinant PAC clones is obtained in pPAC7 because the cloning sites are located within the SacBII gene. We show regulated expression of the CDH3 gene carried as a 132 kb genomic insert cloned into pPAC7, demonstrating that the pPAC7 vector can be used for functional studies of genes in their natural genomic context. Furthermore, the results from the transfection of a range of pPAC7 based constructs into two human cell lines suggest that the transfection efficiencies are not only dependent on construct size. CONCLUSION: The shuttle vector pPAC7 can be used to transfer large genomic constructs into human cells. The genes transferred could potentially contain all long-range regulatory elements, including their endogenous regulatory promoters. Introduction of complete genes in PACs into human cells would potentially allow complementation assays to identify or verify the function of genes affecting cellular phenotypes.

Visualizing stromal cell dynamics in different tumor microenvironments by spinning disk confocal microscopy.

The tumor microenvironment consists of stromal cells and extracellular factors that evolve in parallel with carcinoma cells. To gain insights into the activities of stromal cell populations, we developed and applied multicolor imaging techniques to analyze the behavior of these cells within different tumor microenvironments in the same live mouse. We found that regulatory T-lymphocytes (Tregs) migrated in proximity to blood vessels. Dendritic-like cells, myeloid cells and carcinoma-associated fibroblasts all exhibited higher motility in the microenvironment at the tumor periphery than within the tumor mass. Since oxygen levels differ between tumor microenvironments, we tested if acute hypoxia could account for the differences in cell migration. Direct visualization revealed that Tregs ceased migration under acute systemic hypoxia, whereas myeloid cells continued migrating. In the same mouse and microenvironment, we experimentally subdivided the myeloid cell population and revealed that uptake of fluorescent dextran defined a low-motility subpopulation expressing markers of tumor-promoting, alternatively activated macrophages. In contrast, fluorescent anti-Gr1 antibodies marked myeloid cells patrolling inside tumor vessels and in the stroma. Our techniques allow real-time combinatorial analysis of cell populations based on spatial location, gene expression, behavior and cell surface molecules within intact tumors. The techniques are not limited to investigations in cancer, but could give new insights into cell behavior more broadly in development and disease.

Matrix metalloproteinase 13 is induced in fibroblasts in polyomavirus middle T antigen-driven mammary carcinoma without influencing tumor progression.

Matrix metalloproteinase (MMP) 13 (collagenase 3) is an extracellular matrix remodeling enzyme that is induced in myofibroblasts during the earliest invasive stages of human breast carcinoma, suggesting that it is involved in tumor progression. During progression of mammary carcinomas in the polyoma virus middle T oncogene mouse model (MMTV-PyMT), Mmp13 mRNA was strongly upregulated concurrently with the transition to invasive and metastatic carcinomas. As in human tumors, Mmp13 mRNA was found in myofibroblasts of invasive grade II and III carcinomas, but not in benign grade I and II mammary intraepithelial neoplasias. To determine if MMP13 plays a role in tumor progression, we crossed MMTV-PyMT mice with Mmp13 deficient mice. The absence of MMP13 did not influence tumor growth, vascularization, progression to more advanced tumor stages, or metastasis to the lungs, and the absence of MMP13 was not compensated for by expression of other MMPs or tissue inhibitor of metalloproteinases. However, an increased fraction of thin collagen fibrils was identified in MMTV-PyMT;Mmp13(-/-) compared to MMTV-PyMT;Mmp13(+/+) tumors, showing that collagen metabolism was altered in the absence of MMP13. We conclude that the expression pattern of Mmp13 mRNA in myofibroblasts of invasive carcinomas in the MMTV-PyMT breast cancer model recapitulates the expression pattern observed in human breast cancer. Our results suggest that MMP13 is a marker of carcinoma-associated myofibroblasts of invasive carcinoma, even though it does not make a major contribution to tumor progression in the MMTV-PyMT breast cancer model.