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Colorectal cancer is caused by a sequence of somatic genomic alterations affecting driver genes in core cancer pathways¹. Here, to understand the functional and prognostic impact of cancer-causing somatic mutations, we analysed the whole genomes and transcriptomes of 1,063 primary colorectal cancers in a population-based cohort with long-term follow-up. From the 96 mutated driver genes, 9 were not previously implicated in colorectal cancer and 24 had not been linked to any cancer. Two distinct patterns of pathway co-mutations were observed, timing analyses identified nine early and three late driver gene mutations, and several signatures of colorectal-cancer-specific mutational processes were identified. Mutations in WNT, EGFR and TGFβ pathway genes, the mitochondrial CYB gene and 3 regulatory elements along with 21 copy-number variations and the COSMIC SBS44 signature correlated with survival. Gene expression classification yielded five prognostic subtypes with distinct molecular features, in part explained by underlying genomic alterations. Microsatellite-instable tumours divided into two classes with different levels of hypoxia and infiltration of immune and stromal cells. To our knowledge, this study constitutes the largest integrated genome and transcriptome analysis of colorectal cancer, and interlinks mutations, gene expression and patient outcomes. The identification of prognostic mutations and expression subtypes can guide future efforts to individualize colorectal cancer therapy.

Mutation analysis of circulating tumor DNA (ctDNA) has applications in monitoring of colorectal cancer (CRC) patients for recurrence. Considering the low tumor fraction of ctDNA in cell-free DNA (cfDNA) isolated from blood plasma, the sensitivity of the detection method is important. Here, plasma DNA collected at diagnosis and follow-up from 25 CRC patients was analyzed using a multiplex superRCA mutation detection assay. The assay was also performed on genomic DNA (gDNA) from tumor and normal tissue from 20 of these patients. The lower limit of detection for most sequence variants was in the range of 10⁻⁵, while when analyzing cfDNA from plasma with a typical input of 33 ng, the practical detection limit was ~10⁻⁴ or 0.01% mutant allele frequency (MAF). In 17 of 19 patients with identified hotspot mutations in tumor gDNA, at least one hotspot mutation could be detected in plasma DNA at the time of diagnosis. The MAF increased at subsequent time points in four of the patients who experienced a clinical relapse. Multiplex superRCA analysis of the remaining six patients did not reveal any hotspot mutations. In conclusion, multiplex superRCA assays proved suitable for monitoring CRC patients by analyzing hotspot mutations in cfDNA, and dynamic changes in MAF were observed in patients with clinical relapse.

Background: Cancer immunity is based on the interaction of a multitude of cells in the spatial context of the tumour tissue. Clinically relevant immune signatures are therefore anticipated to fundamentally improve the accuracy in predicting disease progression.

Methods: Through a multiplex in situ analysis we evaluated 15 immune cell classes in 1481 tumour samples. Single-cell and bulk RNAseq data sets were used for functional analysis and validation of prognostic and predictive associations.

Findings: By combining the prognostic information of anti-tumoural CD8+ lymphocytes and tumour supportive CD68+CD163+ macrophages in colorectal cancer we generated a signature of immune activation (SIA). The prognostic impact of SIA was independent of conventional parameters and comparable with the state-of-art immune score. The SIA was also associated with patient survival in oesophageal adenocarcinoma, bladder cancer, lung adenocarcinoma and melanoma, but not in endometrial, ovarian and squamous cell lung carcinoma. We identified CD68+CD163+ macrophages as the major producers of complement C1q, which could serve as a surrogate marker of this macrophage subset. Consequently, the RNA-based version of SIA (ratio of CD8A to C1QA) was predictive for survival in independent RNAseq data sets from these six cancer types. Finally, the CD8A/C1QA mRNA ratio was also predictive for the response to checkpoint inhibitor therapy.

Interpretation: Our findings extend current concepts to procure prognostic information from the tumour immune microenvironment and provide an immune activation signature with high clinical potential in common human cancer types.

Comprehensive and scalable proteomic profiling of plasma samples can improve the screening and diagnosis of cancer patients. Here, the authors use the Olink Proximity Extension Assay technology to characterise the plasma proteomes of 1477 patients across twelve cancer types, and use machine learning to obtain a protein panel for cancer classification. A comprehensive characterization of blood proteome profiles in cancer patients can contribute to a better understanding of the disease etiology, resulting in earlier diagnosis, risk stratification and better monitoring of the different cancer subtypes. Here, we describe the use of next generation protein profiling to explore the proteome signature in blood across patients representing many of the major cancer types. Plasma profiles of 1463 proteins from more than 1400 cancer patients are measured in minute amounts of blood collected at the time of diagnosis and before treatment. An open access Disease Blood Atlas resource allows the exploration of the individual protein profiles in blood collected from the individual cancer patients. We also present studies in which classification models based on machine learning have been used for the identification of a set of proteins associated with each of the analyzed cancers. The implication for cancer precision medicine of next generation plasma profiling is discussed.

The gold standard for diagnosing endometriosis is by laparoscopic visual demonstration of ectopic endometrial lesions outside the uterus, preferably verified by biopsy and microscopical examination. Molecular markers to facilitate the microscopical diagnosis of endometriosis and for distinguishing endometriosis from other benign and malignant lesions are lacking. Our aim was to test and validate an immunohistochemical antibody panel for improved diagnostic accuracy of endometriosis. Both CD10 and HOXA11 have been implicated in regulation of endometrial homeostasis. Here we have analyzed the expression pattern of these two proteins using immunohistochemistry on human tissues in a tissue microarray format. CD10 and HOXA11 expression in endometriosis lesions were compared to expression patterns in a range of normal tissues and in primary- and metastatic lesions of endometrial-, cervical- and ovarian cancer. HOXA11 and CD10 were expressed in 98% and 91% of endometriosis lesions and the combined double-positive expression profile of both HOXA11 and CD10 was highly sensitive for ectopic endometrial tissue (90%). The specificity and sensitivity for this double-positive signature in endometriosis was significantly different from all investigated tissues, cancers and metastases except normal, eutopic endometrial- and cervical mucosa. The combination of HOXA11 and CD10 expression profiles provides a useful tool to identify ectopic endometrial tissue and for distinguishing endometriosis from various types of gynecological malignancies and metastases.

BACKGROUND: Ovarian cancer is the eighth most common cancer among women and has a 5-year survival of only 30-50%. The survival is close to 90% for patients in stage I but only 20% for patients in stage IV. The presently available biomarkers have insufficient sensitivity and specificity for early detection and there is an urgent need to identify novel biomarkers.

METHODS: We employed the Explore PEA technology for high-precision analysis of 1463 plasma proteins and conducted a discovery and replication study using two clinical cohorts of previously untreated patients with benign or malignant ovarian tumours (N = 111 and N = 37).

RESULTS: The discovery analysis identified 32 proteins that had significantly higher levels in malignant cases as compared to benign diagnoses, and for 28 of these, the association was replicated in the second cohort. Multivariate modelling identified three highly accurate models based on 4 to 7 proteins each for separating benign tumours from early-stage and/or late-stage ovarian cancers, all with AUCs above 0.96 in the replication cohort. We also developed a model for separating the early-stage from the late-stage achieving an AUC of 0.81 in the replication cohort. These models were based on eleven proteins in total (ALPP, CXCL8, DPY30, IL6, IL12, KRT19, PAEP, TSPAN1, SIGLEC5, VTCN1, and WFDC2), notably without MUCIN-16. The majority of the associated proteins have been connected to ovarian cancer but not identified as potential biomarkers.

CONCLUSIONS: The results show the ability of using high-precision proteomics for the identification of novel plasma protein biomarker candidates for the early detection of ovarian cancer.

Cancer mortality is exacerbated by late-stage diagnosis. Liquid biopsies based on genomic biomarkers can noninvasively diagnose cancers. However, validation studies have reported similar to 10% sensitivity to detect stage I cancer in a screening population and specific types, such as brain or genitourinary tumors, remain undetectable. We investigated urine and plasma free glycosaminoglycan profiles (GAGomes) as tumor metabolism biomarkers for multi-cancer early detection (MCED) of 14 cancer types using 2,064 samples from 1,260 cancer or healthy subjects. We observed widespread cancer-specific changes in biofluidic GAGomes recapitulated in an in vivo cancer progression model. We developed three machine learning models based on urine (N-urine = 220 cancer vs. 360 healthy) and plasma (N-plasma = 517 vs. 425) GAGomes that can detect any cancer with an area under the receiver operating characteristic curve of 0.83-0.93 with up to 62% sensitivity to stage I disease at 95% specificity. Undetected patients had a 39 to 50% lower risk of death. GAGomes predicted the putative cancer location with 89% accuracy. In a validation study on a screening-like population requiring >= 99% specificity, combined GAGomes predicted any cancer type with poor prognosis within 18 months with 43% sensitivity (21% in stage I; N = 121 and 49 cases). Overall, GAGomes appeared to be powerful MCED metabolic biomarkers, potentially doubling the number of stage I cancers detectable using genomic biomarkers.

We sought to provide a detailed overview of the immune landscape of colorectal cancer in the largest study to date in terms of patient numbers and analyzed immune cell types. We applied a multiplex in situ staining method in combination with an advanced scanning and image analysis pipeline akin to flow cytometry, and analyzed 5968 individual multi-layer images of tissue defining in a total of 39,078,450 cells. We considered the location of immune cells with respect to the stroma, and tumor cell compartment and tumor regions in the central part or the invasive margin. To the best of our knowledge, this study is the first comprehensive spatial description of the immune landscape in colorectal cancer using a large population-based cohort and a multiplex immune cell identification.<br>While the clinical importance of CD8+ and CD3+ cells in colorectal cancer (CRC) is well established, the impact of other immune cell subsets is less well described. We sought to provide a detailed overview of the immune landscape of CRC in the largest study to date in terms of patient numbers and in situ analyzed immune cell types. Tissue microarrays from 536 patients were stained using multiplexed immunofluorescence panels, and fifteen immune cell subclasses, representing adaptive and innate immunity, were analyzed. Overall, therapy-naive CRC patients clustered into an 'inflamed' and a 'desert' group. Most T cell subsets and M2 macrophages were enriched in the right colon (p-values 0.046-0.004), while pDC cells were in the rectum (p = 0.008). Elderly patients had higher infiltration of M2 macrophages (p = 0.024). CD8+ cells were linked to improved survival in colon cancer stages I-III (q = 0.014), while CD4+ cells had the strongest impact on overall survival in metastatic CRC (q = 0.031). Finally, we demonstrated repopulation of the immune infiltrate in rectal tumors post radiation, following an initial radiation-induced depletion. This study provides a detailed analysis of the in situ immune landscape of CRC paving the way for better diagnostics and providing hints to better target the immune microenvironment.

Background: The development of a reactive tumour stroma is a hallmark of tumour progression and pronounced tumour stroma is generally considered to be associated with clinical aggressiveness. The variability between tumour types regarding stroma fraction, and its prognosis associations, have not been systematically analysed.

Methods: Using an objective machine-learning method we quantified the tumour stroma in 16 solid cancer types from 2732 patients, representing retrospective tissue collections of surgically resected primary tumours. Image analysis performed tissue segmentation into stromal and epithelial compartment based on pan-cytokeratin staining and autofluorescence patterns.

Findings: The stroma fraction was highly variable within and across the tumour types, with kidney cancer showing the lowest and pancreato-biliary type periampullary cancer showing the highest stroma proportion (median 19% and 73% respectively). Adjusted Cox regression models revealed both positive (pancreato-biliary type periampullary cancer and oestrogen negative breast cancer, HR(95%CI)=0.56(0.34-0.92) and HR (95%CI)=0.41(0.17-0.98) respectively) and negative (intestinal type periampullary cancer, HR(95%CI)=3.59 (1.49-8.62)) associations of the tumour stroma fraction with survival.

Interpretation: Our study provides an objective quantification of the tumour stroma fraction across major types of solid cancer. Findings strongly argue against the commonly promoted view of a general associations between high stroma abundance and poor prognosis. The results also suggest that full exploitation of the prognostic potential of tumour stroma requires analyses that go beyond determination of stroma abundance.

Background: Survival of metastatic colorectal cancer (mCRC) patients has improved, but mainly for trial patients. New predictive and prognostic biomarkers validated in the general mCRC population are needed. Caudal-type homeobox 2 (CDX2) is an intestine-specific transcription factor with potential prognostic and predictive effect, but the importance in mCRC has not been fully investigated. Methods: Immunohistochemistry analysis of CDX2 was performed in a Scandinavian population-based cohort of mCRC (n = 796). Frequency, clinical and tumor characteristics, response rate, progression-free survival, and overall survival (OS) were estimated. Results: Loss of CDX2 expression was found in 87 (19%) of 452 stained cases, in 53% if BRAF mutated (BRAFmut) and in 9% if KRAS mutated (KRASmut). CDX2 loss was associated with microsatellite instability, BRAFmut, and poor differentiation and inversely associated with KRASmut. Patients with CDX2 loss received less first-line (53 vs. 64%, p = 0.050) and second-line (23 vs. 39%, p = 0.006) chemotherapy and secondary surgery (1 vs. 9%, p = 0.019). Median progression-free survival and OS for patients given first-line combination chemotherapy was 4 and 10 months if CDX2 loss vs. 9 and 24 months if CDX2 expressed (p = 0.001, p < 0.001). Immediate progression on first-line combination chemotherapy was seen in 35% of patients with CDX2 loss vs. 10% if CDX2 expressed (p = 0.003). Median OS in patients with BRAFmut or KRASmut and CDX2 expressed in tumor (both 21 months) was comparable to wild-type patients (27 months). However, if CDX2 loss, median OS was only 8 and 11 months in BRAFmut and KRASmut cases, respectively, and 10 months in double wild-type patients. In multivariate analysis, CDX2 loss (hazard ratio: 1.50, p = 0.027) and BRAFmut (hazard ratio: 1.62, p = 0.012) were independent poor prognostic markers for OS. Conclusion: In a population-based cohort of mCRC patients, CDX2 loss is an independent poor prognostic marker. Expression of CDX2 defines a new subgroup of BRAFmut cases with a much better prognosis. Loss of CDX2 defines a small group of KRASmut cases with a worse prognosis. Patients with CDX2 loss receive less palliative chemotherapy with less benefit and rarely reach secondary surgery.