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Abstract In the spectrum of colorectal tumors, microsatellite-stable (MSS) tumors with DNA polymerase ε (POLE) mutations exhibit a hypermutated profile, holding the potential to respond to immunotherapy similarly to their microsatellite-instable (MSI) counterparts. Yet, due to their rarity and the associated testing costs, systematic screening for these mutations is not commonly pursued. Notably, the histopathological phenotype resulting from POLE mutations is theorized to resemble that of MSI. This resemblance not only could facilitate their detection by a transformer-based Deep Learning (DL) system trained on MSI pathology slides, but also indicates the possibility for MSS patients with POLE mutations to access enhanced treatment options, which might otherwise be overlooked. To harness this potential, we trained a Deep Learning classifier on a large dataset with the ground truth for microsatellite status and subsequently validated its capabilities for MSI and POLE detection across three external cohorts. Our model accurately identified MSI status in both the internal and external resection cohorts using pathology images alone. Notably, with a classification threshold of 0.5, over 75% of POLE driver mutant patients in the external resection cohorts were flagged as “positive” by a DL system trained on MSI status. In a clinical setting, deploying this DL model as a preliminary screening tool could facilitate the efficient identification of clinically relevant MSI and POLE mutations in colorectal tumors, in one go.

Abstract The development of deep learning (DL) models to predict the consensus molecular subtypes (CMS) from histopathology images (imCMS) is a promising and cost-effective strategy to support patient stratification. Here, we investigate whether imCMS calls generated from whole slide histopathology images (WSIs) of rectal cancer (RC) pre-treatment biopsies are associated with pathological complete response (pCR) to neoadjuvant long course chemoradiotherapy (LCRT) with single agent fluoropyrimidine. DL models were trained to classify WSIs of colorectal cancers stained with hematoxylin and eosin into one of the four CMS classes using a multi-centric dataset of resection and biopsy specimens (n = 1057 WSIs) with paired transcriptional data. Classifiers were tested on a held out RC biopsy cohort (ARISTOTLE) and correlated with pCR to LCRT in an independent dataset merging two RC cohorts (ARISTOTLE, n = 114 and SALZBURG, n = 55 patients). DL models predicted CMS with high classification performance in multiple comparative analyses. In the independent cohorts (ARISTOTLE, SALZBURG), cases with WSIs classified as imCMS1 had a significantly higher likelihood of achieving pCR (OR = 2.69, 95% CI 1.01–7.17, p = 0.048). Conversely, imCMS4 was associated with lack of pCR (OR = 0.25, 95% CI 0.07–0.88, p = 0.031). Classification maps demonstrated pathologist-interpretable associations with high stromal content in imCMS4 cases, associated with poor outcome. No significant association was found in imCMS2 or imCMS3. imCMS classification of pre-treatment biopsies is a fast and inexpensive solution to identify patient groups that could benefit from neoadjuvant LCRT. The significant associations between imCMS1/imCMS4 with pCR suggest the existence of predictive morphological features that could enhance standard pathological assessment.

Summary: Background: It is uncertain which biological features underpin the response of rectal cancer (RC) to radiotherapy. No biomarker is currently in clinical use to select patients for treatment modifications. Methods: We identified two cohorts of patients (total N = 249) with RC treated with neoadjuvant radiotherapy (45Gy/25) plus fluoropyrimidine. This discovery set included 57 cases with pathological complete response (pCR) to chemoradiotherapy (23%). Pre-treatment cancer biopsies were assessed using transcriptome-wide mRNA expression and targeted DNA sequencing for copy number and driver mutations. Biological candidate and machine learning (ML) approaches were used to identify predictors of pCR to radiotherapy independent of tumour stage. Findings were assessed in 107 cases from an independent validation set (GSE87211). Findings: Three gene expression sets showed significant independent associations with pCR: Fibroblast-TGFβ Response Signature (F-TBRS) with radioresistance; and cytotoxic lymphocyte (CL) expression signature and consensus molecular subtype CMS1 with radiosensitivity. These associations were replicated in the validation cohort. In parallel, a gradient boosting machine model comprising the expression of 33 genes generated in the discovery cohort showed high performance in GSE87211 with 90% sensitivity, 86% specificity. Biological and ML signatures indicated similar mechanisms underlying radiation response, and showed better AUC and p-values than published transcriptomic signatures of radiation response in RC. Interpretation: RCs responding completely to chemoradiotherapy (CRT) have biological characteristics of immune response and absence of immune inhibitory TGFβ signalling. These tumours may be identified with a potential biomarker based on a 33 gene expression signature. This could help select patients likely to respond to treatment with a primary radiotherapy approach as for anal cancer. Conversely, those with predicted radioresistance may be candidates for clinical trials evaluating addition of immune-oncology agents and stromal TGFβ signalling inhibition. Funding: The Stratification in Colorectal Cancer Consortium (S:CORT) was funded by the Medical Research Council and Cancer Research UK (MR/M016587/1).

Abstract Tumor deposits (TDs) are discontinuous tumor spread in the mesocolon/mesorectum which is found in approximately 20% of colorectal cancer (CRC) and negatively affects survival. We have a history of repeated revisions on TD definition and categorization in the tumor‐node‐metastasis (TNM) system leading to stage migration. Since 1997, TDs have been categorized as T or N factors depending on their size (TNM5) or contour (TNM6). In 2009, TNM7 provided the category of N1c for TDs in a case without positive lymph nodes (LNs), which is also used in TNM8. However, increasing evidence suggests that these revisions are suboptimal and only “partially” successful. Specifically, the N1c rule is certainly useful for oncologists who are having difficulty with TDs in a case with no positive LNs. However, it has failed to maximize the value of the TNM system because of the underused prognostic information of individual TDs. Recently, the potential value of an alternative staging method has been highlighted in several studies using the “counting method.” For this method, all nodular type TDs are individually counted together with positive LNs to derive the final pN, yielding a prognostic and diagnostic value that is superior to existing TNM systems. The TNM system has long stuck to the origin of TDs in providing its categorization, but it is time to make way for alternative options and initiate an international discussion on optimal treatment of TDs in tumor staging; otherwise, a proportion of patients end up missing an opportunity to receive the optimal adjuvant treatment.

Abstract It is increasingly being recognised that changes in the gut microbiome have either a causative or associative relationship with colorectal cancer (CRC). However, most of this research has been carried out in a small number of developed countries with high CRC incidence. It is unknown if lower incidence countries such as India have similar microbial associations. Having previously established protocols to facilitate microbiome research in regions with developing research infrastructure, we have now collected and sequenced microbial samples from a larger cohort study of 46 Indian CRC patients and 43 healthy volunteers. When comparing to previous global collections, these samples resemble other Asian samples, with relatively high levels of Prevotella. Predicting cancer status between cohorts shows good concordance. When compared to a previous collection of Indian CRC patients, there was similar concordance, despite different sequencing technologies between cohorts. These results show that there does seem to be a global CRC microbiome, and that some inference between studies is reasonable. However, we also demonstrate that there is definite regional variation, with more similarities between location-matched comparisons. This emphasises the importance of developing protocols and advancing infrastructure to allow as many countries as possible to contribute to microbiome studies of their own populations.

Abstract Background Lynch Syndrome (LS) is an inherited cancer predisposition syndrome defined by pathogenic variants in the mismatch repair (MMR) or EPCAM genes. In the United Kingdom, people with LS are advised to undergo biennial colonoscopy from as early as 25 until 75 years of age to mitigate a high lifetime colorectal cancer (CRC) risk, though the consideration of additional surveillance intervention(s) through the application of non-invasive diagnostic devices has yet to be longitudinally observed in LS patients. In this study, we will examine the role of annual faecal immunochemical testing (FIT) alongside biennial colonoscopy for CRC surveillance in people with LS. Methods/design In this single-arm, prospective, non-randomised study, 400 LS patients will be recruited across 11 National Health Service (NHS) Trusts throughout the United Kingdom. Study inclusion requires a LS diagnosis, between 25 and 73 years old, and a routine surveillance colonoscopy scheduled during the recruitment period. Eligible patients will receive a baseline OC-Sensor™ FIT kit ahead of their colonoscopy, and annually for 3 years thereafter. A pre-paid envelope addressed to the central lab will be included within all patient mailings for the return of FIT kits and relevant study documents. A questionnaire assessing attitudes and perception of FIT will also be included at baseline. All study samples received by the central lab will be assayed on an OC-Sensor™ PLEDIA Analyser. Patients with FIT results of ≥6 μg of Haemoglobin per gram of faeces (f-Hb) at Years 1 and/or 3 will be referred for colonoscopy via an urgent colonoscopy triage pathway. 16S rRNA gene V4 amplicon sequencing will be carried out on residual faecal DNA of eligible archived FIT samples to characterise the faecal microbiome. Discussion FIT may have clinical utility alongside colonoscopic surveillance in people with LS. We have designed a longitudinal study to examine the efficacy of FIT as a non-invasive modality. Potential limitations of this method will be assessed, including false negative or false positive FIT results related to specific morphological features of LS neoplasia or the presence of post-resection anastomotic inflammation. The potential for additional colonoscopies in a subset of participants may also impact on colonoscopic resources and patient acceptability. Trial registration Trial Registration: ISRCTN, ISRCTN15740250 . Registered 13 July 2021.

The Grb2 splice variant Grb3-3 results in decreased activation of the Ras-MAPK pathway due competition with Grb2 for SOS binding, with reduced Grb3-3 levels observed in tumour tissue suggestive of a mechanism for enhancing Ras pathway activity.

Background: Complex trials with innovative designs are becoming increasingly common and offer the potential to improve patient outcomes in a shorter time frame. There is evidence that patients with colorectal cancer fall into different subgroups with varying responsiveness to therapy, and that this variation is linked to genetic biomarkers. To the best of our knowledge, FOCUS4 was the first molecularly stratified trial in metastatic colorectal cancer and remains one of the first umbrella trial designs to be launched globally. Objectives: To identify novel therapies that improve disease control within the molecular subgroup of metastatic colorectal cancer in which the novel therapies were expected to be most effective. Design: This was a Phase II/III molecularly stratified umbrella trial that used adaptive statistical methodology to decide which subtrial should close early; new subtrials were added as protocol amendments. Setting: The maintenance setting following 16 weeks of first-line combination chemotherapy. Participants: Patients with newly diagnosed metastatic colorectal cancer were registered, and central laboratory testing was used to stratify their tumour into molecular subtypes. Following 16 weeks of first-line therapy, patients with stable or responding disease were eligible for randomisation into either a molecularly stratified subtrial or the non-stratified FOCUS4-N trial. Interventions: Of the 20 drug combinations that were explored for inclusion in the platform trial, three molecularly targeted subtrials were activated: FOCUS4-B (PIK3CA mutation or PTEN overexpression) – aspirin versus placebo; FOCUS4-C (TP53 and RAS mutation) – adavosertib (AstraZeneca Ltd, Cambridge, UK) versus active monitoring; and FOCUS4-D (BRAF-PIK3CA-RAS wild type) – AZD8931 versus placebo. A non-stratified subtrial was also carried out: FOCUS4-N – capecitabine versus active monitoring. Main outcome measures: The main outcome measure was progression-free survival from the time of randomisation to progression, comparing the intervention with active monitoring/placebo. Toxicity and overall survival data were collected in all randomised patients, and quality of life (using EuroQol-5 Dimensions) data were collected in FOCUS4-N only. Results: Between January 2014 and October 2020, 1434 patients were registered from 88 hospitals in the UK. Successful biomarker testing was completed in 1291 out of 1382 samples (93%), and 908 out of 1315 patients (69%) completing 16 weeks of first-line therapy were eligible for randomisation, with 361 randomly allocated to a subtrial. FOCUS4-B evaluated aspirin versus placebo in the PIK3CA-mutant/PTEN-loss subgroup, but recruited only six patients, so was closed for futility. FOCUS4-C evaluated adavosertib versus active monitoring in 67 patients in the RAS + TP53 double-mutant subgroup and met its primary end point, showing an improvement in progression-free survival (median 3.61 vs. 1.87 months; hazard ratio 0.35, 95% confidence interval 0.18 to 0.68; p = 0022). FOCUS4-D evaluated AZD8931 in 32 patients in the BRAF-PIK3CA-RAS wild-type subgroup and showed no benefit, so was discontinued after the first interim analysis. FOCUS4-N evaluated capecitabine monotherapy versus active monitoring in 254 patients and met its primary end point, showing improvement in progression-free survival (hazard ratio 0.40, 95% confidence interval 0.21 to 0.75; p

The composition and cell-to-cell interactions of the peripheral immune compartment are known to influence outcomes in multiple disease entities, both neoplastic and otherwise. There is an ongoing search for a reliable biomarker in the peripheral immune compartment that can predict outcomes in colorectal cancer, given its high mortality rates. The neutrophil:lymphocyte ratio (NLR) has been suggested as one such marker, given its accessibility. This review discusses the current evidence behind the use of the NLR in predicting different aspects of colorectal cancer (CRC) behaviour, from survival to recurrence, metastasis, tumour biology, response to therapy and CRC complications. We also discuss the debate in the literature surrounding the use of other peripheral immune compartment biomarkers compared with the NLR for this purpose and ideas for future research.

Abstract Background The incidence of colorectal cancer (CRC) is increasing in developing countries, yet limited research on the CRC- associated microbiota has been conducted in these areas, in part due to scarce resources, facilities, and the difficulty of fresh or frozen stool storage/transport. Here, we aimed (1) to establish a broad representation of diverse developing countries (Argentina, Chile, India, and Vietnam); (2) to validate a ‘resource-light’ sample-collection protocol translatable in these settings using guaiac faecal occult blood test (gFOBT) cards stored and, importantly, shipped internationally at room temperature; (3) to perform initial profiling of the collective CRC-associated microbiome of these developing countries; and (4) to compare this quantitatively with established CRC biomarkers from developed countries. Methods We assessed the effect of international storage and transport at room temperature by replicating gFOBT from five UK volunteers, storing two in the UK, and sending replicates to institutes in the four countries. Next, to determine the effect of prolonged UK storage, DNA extraction replicates for a subset of samples were performed up to 252 days apart. To profile the CRC-associated microbiome of developing countries, gFOBT were collected from 41 treatment-naïve CRC patients and 40 non-CRC controls from across the four institutes, and V4 16S rRNA gene sequencing was performed. Finally, we constructed a random forest (RF) model that was trained and tested against existing datasets from developed countries. Results The microbiome was stably assayed when samples were stored/transported at room temperature and after prolonged UK storage. Large-scale microbiome structure was separated by country and continent, with a smaller effect from CRC. Importantly, the RF model performed similarly to models trained using external datasets and identified similar taxa of importance (Parvimonas, Peptostreptococcus, Fusobacterium, Alistipes, and Escherichia). Conclusions This study demonstrates that gFOBT, stored and transported at room temperature, represents a suitable method of faecal sample collection for amplicon-based microbiome biomarkers in developing countries and suggests a CRC-faecal microbiome association that is consistent between developed and developing countries.

Abstract Background Pre-operative chemoradiotherapy (CRT) for MRI-defined, locally advanced rectal cancer is primarily intended to reduce local recurrence rates by downstaging tumours, enabling an improved likelihood of curative resection. However, in a subset of patients complete tumour regression occurs implying that no viable tumour is present within the surgical specimen. This raises the possibility that surgery may have been avoided. It is also recognised that response to CRT is a key determinant of prognosis. Recent radiological advances enable this response to be assessed pre-operatively using the MRI tumour regression grade (mrTRG). Potentially, this allows modification of the baseline MRI-derived treatment strategy. Hence, in a ‘good’ mrTRG responder, with little or no evidence of tumour, surgery may be deferred. Conversely, a ‘poor response’ identifies an adverse prognostic group which may benefit from additional pre-operative therapy. Methods/design TRIGGER is a multicentre, open, interventional, randomised control feasibility study with an embedded phase III design. Patients with MRI-defined, locally advanced rectal adenocarcinoma deemed to require CRT will be eligible for recruitment. During CRT, patients will be randomised (1:2) between conventional management, according to baseline MRI, versus mrTRG-directed management. The primary endpoint of the feasibility phase is to assess the rate of patient recruitment and randomisation. Secondary endpoints include the rate of unit recruitment, acute drug toxicity, reproducibility of mrTRG reporting, surgical morbidity, pathological circumferential resection margin involvement, pathology regression grade, residual tumour cell density and surgical/specimen quality rates. The phase III trial will focus on long-term safety, regrowth rates, oncological survival analysis, quality of life and health economics analysis. Discussion The TRIGGER trial aims to determine whether patients with locally advanced rectal cancer can be recruited and subsequently randomised into a control trial that offers MRI-directed patient management according to radiological response to CRT (mrTRG). The feasibility study will inform a phase III trial design investigating stratified treatment of good and poor responders according to 3-year disease-free survival, colostomy-free survival as well as an increase in cases managed without a major resection. Trial registration ClinicalTrials.gov, ID: NCT02704520 . Registered on 5 February 2016.

Background: Robotic rectal cancer surgery is gaining popularity, but there are limited data about its safety and efficacy. Objective: To undertake an evaluation of robotic compared with laparoscopic rectal cancer surgery to determine its safety, efficacy and cost-effectiveness. Design: This was a multicentre, randomised trial comparing robotic with laparoscopic rectal resection in patients with rectal adenocarcinoma. Setting: The study was conducted at 26 sites across 10 countries and involved 40 surgeons. Participants: The study involved 471 patients with rectal adenocarcinoma. Recruitment took place from 7 January 2011 to 30 September 2014 with final follow-up on 16 June 2015. Interventions: Robotic and laparoscopic rectal cancer resections were performed by high anterior resection, low anterior resection or abdominoperineal resection. There were 237 patients randomised to robotic and 234 to laparoscopic surgery. Follow-up was at 30 days, at 6 months and annually until 3 years after surgery. Main outcome measures: The primary outcome was conversion to laparotomy. Secondary end points included intra- and postoperative complications, pathological outcomes, quality of life (QoL) [measured using the Short Form questionnaire-36 items version 2 (SF-36v2) and the Multidimensional Fatigue Inventory-20 (MFI-20)], bladder and sexual dysfunction [measured using the International Prostatic Symptom Score (I-PSS), the International Index of Erectile Function (IIEF) and the Female Sexual Function Index (FSFI)], and oncological outcomes. An economic evaluation considered the costs of robotic and laparoscopic surgery, including primary and secondary care costs up to 6 months post operation. Results: Among 471 randomised patients [mean age 64.9 years, standard deviation (SD) 11.0 years; 320 (67.9%) men], 466 (98.9%) patients completed the study. Data were analysed on an intention-to-treat basis. The overall rate of conversion to laparotomy was 10.1% and occurred in 19 (8.1%) patients in the robotic-assisted group and in 28 (12.2%) patients in the conventional laparoscopic group {unadjusted risk difference 4.12% [95% confidence interval (CI) –1.35% to 9.59%], adjusted odds ratio 0.61 [95% CI 0.31 to –1.21]; p = 0.16}. Of the nine prespecified secondary end points, including circumferential resection margin positivity, intraoperative complications, postoperative complications, plane of surgery, 30-day mortality and bladder and sexual dysfunction, none showed a statistically significant difference between the groups. No difference between the treatment groups was observed for longer-term outcomes, disease-free and overall survival (OS). Males were at a greater risk of local recurrence than females and had worse OS rates. The costs of robotic and laparoscopic surgery, excluding capital costs, were £11,853 (SD £2940) and £10,874 (SD £2676) respectively. Conclusions: There is insufficient evidence to conclude that robotic rectal surgery compared with laparoscopic rectal surgery reduces the risk of conversion to laparotomy. There were no statistically significant differences in resection margin positivity, complication rates or QoL at 6 months between the treatment groups. Robotic rectal cancer surgery was on average £980 more expensive than laparoscopic surgery, even when the acquisition and maintenance costs for the robot were excluded. Future work: The lower rate of conversion to laparotomy in males undergoing robotic rectal cancer surgery deserves further investigation. The introduction of new robotic systems into the market may alter the cost-effectiveness of robotic rectal cancer surgery. Trial registration: Current Controlled Trials ISRCTN80500123. Funding: This project was funded by the Efficacy and Mechanism Evaluation (EME) programme, a Medical Research Council and National Institute for Health Research (NIHR) partnership, with contributions from the Chief Scientist Office, Scottish Government Health and Social Care Directorate, the Health and Care Research Wales and the Health and Social Care Research and Development Division, Public Health Agency in Northern Ireland. The funders of the study had no role in the design and conduct of the study; collection, management, analysis and interpretation of the data; and preparation, review or approval of the manuscript or the decision to submit for publication. The project will be published in full in Efficacy and Mechanism Evaluation; Vol. 6, No. 10. See the NIHR Journals Library website for further project information. Philip Quirke and Nicholas West were supported by Yorkshire Cancer Research Campaign and the MRC Bioinformatics initiative. David Jayne was supported by a NIHR Research Professorship.

Background: 5-aminolevulinic acid (5-ALA) is used for fluorescence diagnosis (FD) in neurological, gynaecological and urological malignancies. The Medical Research Council/Efficacy and Mechanism Evaluation (EME) programme/National Institute for Health Research’s Next Generation intraoperative Lymph node staging for Stratified colon cancer surgery (GLiSten) study investigated its use to predict lymph node (LN)-positive disease in colon cancer as an aid to stratified surgery. Objectives: The primary objective was to optimise the dose of oral 5-ALA for intraoperative FD of metastatic LNs in colon cancer. Secondary objectives included standardisation of pre-operative computerised tomography (CT) LN reporting, intraoperative fluorescence detection, surgical resection with D3 lymphadenectomy and histopathological examination of resected specimens. Design: This was a feasibility study to determine optimal strategies for 5-ALA positive LN detection. Patients with locally advanced disease identified using the Fluoropyrimidine, Oxaliplatin and Targeted-Receptor pre-Operative Therapy for patients with high-risk, operable colon cancer (FOxTROT) criteria were recruited from two sites between October 2013 and June 2015. Cohort 1 received 20 mg/kg and cohort 2 received 30 mg/kg of oral 5-ALA, 1–6 hours preoperatively. Laparoscopic assessment of fluorescence was performed using the Storz D-Light system (KARL STORZ GmbH & Co. KG; Tuttlingen, Germany), with marking of fluorescent LNs, followed by oncological resection. The specimen was subjected to histological analysis with step sectioning of marked fluorescent LNs. Progression to an evaluation phase using the optimal dosing schedule was dependent on positively identifying at least 2 out of 10 patients with metastatic LN disease in either cohort. Results: A total of 44 patients were recruited with a male to female ratio of 26 : 18 and a mean age of 71 years (range 52–88 years). Cohort 1 consisted of 18 patients, of whom six had fluorescent primary cancers and three of these had fluorescent LNs. One out of 10 patients with metastatic LN disease had a fluorescent involved LN. Cohort 2 consisted of 26 patients, of whom eight had fluorescent primary cancers and four of these had fluorescent LNs. None of the fluorescent LNs contained disease in this cohort. No serious adverse events (SAEs) occurred but two mild, self-limiting, photosensitivity reactions were observed in cohort 2. The sensitivity and specificity for 5-ALA detection of LN-positive disease were: cohort 1 11.1%, 75%; and cohort 2 0%, 75%. Limitations: This was a feasibility study exploring the use of 5-ALA for LN disease in a select cohort of patients with advanced colorectal cancer. The study population was small and generalisation to other cancers is not possible. The study was limited by the ability to determine LN-positive patients on the basis of pre-operative CT staging, which is often inaccurate, resulting in our cohorts containing several patients without LN disease. Conclusions: 5-ALA fluorescent diagnosis has poor sensitivity for discriminating LN-positive colon cancer. Its use as an aid to stratified colon cancer surgery is not supported. No SAEs were observed, suggesting that photosensitisers may be useful for intraoperative FD. Future work: 5-ALA has poor sensitivity for detecting LN metastases and cannot be recommended for intraoperative staging. Other, more sensitive fluorescent probes are required if this strategy is to be used. Study registration: Current Controlled Trials ISRCTN79949827 and EudraCT number 2012–002623–15. Funding details: This project was funded by the EME programme, a Medical Research Council and National Institute for Health Research partnership.

KRAS mutation status is established as a predictive biomarker of benefit from anti-EGFr therapies. Mutations are normally assessed using DNA extracted from one formalin-fixed, paraffin-embedded (FFPE) tumor block. We assessed heterogeneity of KRAS and BRAF mutation status intra-tumorally (multiple blocks from the same primary tumor). We also investigated the utility and efficiency of genotyping a ‘DNA cocktail’ prepared from multiple blocks. We studied 68 consenting patients in two randomized clinical trials. DNA was extracted, from ≥2 primary tumor FFPE blocks per patient. DNA was genotyped by pyrosequencing for KRAS codons 12, 13 and 61 and BRAF codon 600. In patients with heterogeneous mutation status, DNA cocktails were prepared and genotyped. Among 69 primary tumors in 68 patients, 7 (10.1%) showed intratumoral heterogeneity; 5 (7.2%) at KRAS codons 12, 13 and 2 (2.9%) at BRAF codon 600. In patients displaying heterogeneity, the relevant KRAS or BRAF mutation was also identified in ‘DNA cocktail’ samples when including DNA from mutant and wild-type blocks. Heterogeneity is uncommon but not insignificant. Testing DNA from a single block will wrongly assign wild-type status to 10% patients. Testing more than one block, or preferably preparation of a ‘DNA cocktail’ from two or more tumor blocks, improves mutation detection at minimal extra cost.

There is variability in clinical outcome for patients with apparently the same stage colorectal cancer (CRC). Single nucleotide polymorphisms (SNPs) mapping to chromosomes 1q41, 3q26.2, 6p21, 8q23.3, 8q24.21, 10p14, 11q13, 11q23.1, 12q13.13, 14q22, 14q22.2, 15q13.3, 16q22.1, 18q21.1, 19q13.11, 20p12, 20p12.3, 20q13.33 and Xp22 have robustly been shown to be associated with the risk of developing CRC. Since germline variation can also influence patient outcome the relationship between these SNPs and patient survivorship from CRC was examined.All enrolled into the National Study of Colorectal Cancer Genetics (NSCCG) were genotyped for 1q41, 3q26.2, 6p21, 8q23.3, 8q24.21, 10p14, 11q13, 11q23.1, 12q13.13, 14q22, 14q22.2, 15q13.3, 16q22.1, 18q21.1, 19q13.11, 20p12, 20p12.3, 20q13.33 and xp22 SNPs. Linking this information to the National Cancer Data Repository allowed patient genotype to be related to survival.The linked dataset consisted of 4,327 individuals. 14q22.22 genotype defined by the SNP rs4444235 showed a significant association with overall survival. Specifically, the C allele was associated with poorer observed survival (per allele hazard ratio 1.13, 95% confidence interval 1.05-1.22, P = 0.0015).The CRC susceptibility SNP rs4444235 also appears to exert an influence in modulating patient survival and warrants further evaluation as a potential prognostic marker.

Colorectal cancer (CRC) is one of the leading causes of cancer mortality. Metastasis remains the primary cause of CRC death. Predicting the possibility of metastatic relapse in early-stage CRC is of paramount importance to target therapy for patients who really need it and spare those with low-potential of metastasis. Ninety-six stage II CRC cases were stratified using high-resolution array comparative genomic hybridization (aCGH) data based on a predictive survival algorithm and supervised clustering. All genes included within the resultant copy number aberrations were each interrogated independently at mRNA level using CRC expression datasets available from public repositories, which included 1820 colon cancers, and 167 normal colon tissues. Reduced mRNA expression driven by copy number losses and increased expression driven by copy number gains revealed 42 altered transcripts (29 reduced and 13 increased transcripts) associated with metastatic relapse, short disease-free or overall survival, and/or epithelial to mesenchymal transition (EMT). Resultant genes were classified based on gene ontology (GO), which identified four functional enrichment groups involved in growth regulation, genomic integrity, metabolism, and signal transduction pathways. The identified 42 genes may be useful for predicting metastatic relapse in stage II CRC. Further studies are necessary to validate these findings.

Flat adenomas are a subgroup of colorectal adenomas that have been associated with a distinct biology and a more aggressive clinical behavior compared to their polypoid counterparts. In the present study, we aimed to compare the mutation spectrum of 14 cancer genes, between these two phenotypes.A consecutive series of 106 flat and 93 polypoid adenomas was analyzed retrospectively for frequently occurring mutations in "hot spot" regions of KRAS, BRAF, PIK3CA and NRAS, as well as selected mutations in CTNNB1 (β-catenin), EGFR, FBXW7 (CDC4), PTEN, STK11, MAP2K4, SMAD4, PIK3R1 and PDGFRA using a high-throughput genotyping technique. Additionally, APC was analyzed using direct sequencing.APC mutations were more frequent in polypoid adenomas compared to flat adenomas (48.5% versus 30.3%, respectively, p = 0.02). Mutations in KRAS, BRAF, NRAS, FBXW7 and CTNNB1 showed similar frequencies in both phenotypes. Between the different subtypes of flat adenomas (0-IIa, LST-F and LST-G) no differences were observed for any of the investigated genes.The lower APC mutation rate in flat adenomas compared to polypoid adenomas suggests that disruption of the Wnt-pathway may occur via different mechanisms in these two phenotypes. Furthermore, in contrast to previous observations our results in this large well-defined sample set indicate that there is no significant association between the different morphological phenotypes and mutations in key genes of the RAS-RAF-MAPK pathway.

Background: Obtaining ground truth for pathological images is essential for various experiments, especially for training and testing image analysis algorithms. However, obtaining pathologist input is often difficult, time consuming and expensive. This leads to algorithms being over-fitted to small datasets, and inappropriate validation, which causes poor performance on real world data. There is a great need to gather data from pathologists in a simple and efficient manner, in order to maximise the amount of data obtained. Methods: We present a lightweight, web-based HTML5 system for administering and participating in data collection experiments. The system is designed for rapid input with minimal effort, and can be accessed from anywhere in the world with a reliable internet connection. Results: We present two case studies that use the system to assess how limitations on fields of view affect pathologist agreement, and to what extent poorly stained slides affect judgement. In both cases, the system collects pathologist scores at a rate of less than two seconds per image. Conclusions: The system has multiple potential applications in pathology and other domains.

Background: The Tissue Microarray Data Exchange Specification (TMA DES) is an eXtensible Markup Language (XML) specification for encoding TMA experiment data in a machine-readable format that is also human readable. TMA DES defines Common Data Elements (CDEs) that form a basic vocabulary for describing TMA data. TMA data are routinely subjected to univariate and multivariate statistical analysis to determine differences or similarities between pathologically distinct groups of tumors for one or more markers or between markers for different groups. Such statistical analysis tests include the t-test, ANOVA, Chi-square, Mann-Whitney U, and Kruskal-Wallis tests. All these generate output that needs to be recorded and stored with TMA data. Materials and Methods: We propose extending the TMA DES to include syntactic and semantic definitions of CDEs for describing the results of statistical analyses performed upon TMA DES data. These CDEs are described in this paper and it is illustrated how they can be added to the TMA DES. We created a Document Type Definition (DTD) file defining the syntax for these CDEs, and a set of ISO 11179 entries providing semantic definitions for them. We describe how we wrote a program in R that read TMA DES data from an XML file, performed statistical analyses on that data, and created a new XML file containing both the original XML data and CDEs representing the results of our analyses. This XML file was submitted to XML parsers in order to confirm that they conformed to the syntax defined in our extended DTD file. TMA DES XML files with deliberately introduced errors were also parsed in order to verify that our new DTD file could perform error checking. Finally, we also validated an existing TMA DES XML file against our DTD file in order to demonstrate the backward compatibility of our DTD. Results: Our experiments demonstrated the encoding of analysis results using our proposed CDEs. We used XML parsers to confirm that these XML data were syntactically correct and conformed to the rules specified in our extended TMA DES DTD. We also demonstrated that this extended DTD was capable of being used to successfully perform error checking, and was backward compatible with pre-existing TMA DES data which did not use our new CDEs. Conclusions: The TMA DES allows Tissue Microarray data to be shared. A variety of statistical tests are used to analyze such data. We have proposed a set of CDEs as an extension to the TMA DES which can be used to annotate TMA DES data with the results of statistical analyses performed on that data. We performed experiments which demonstrated the usage of TMA DES data containing our proposed CDEs.

Background: Tissue MicroArrays (TMAs) are a high throughput technology for rapid analysis of protein expression across hundreds of patient samples. Often, data relating to TMAs is specific to the clinical trial or experiment it is being used for, and not interoperable. The Tissue Microarray Data Exchange Specification (TMA DES) is a set of eXtensible Markup Language (XML)-based protocols for storing and sharing digitized Tissue Microarray data. XML data are enclosed by named tags which serve as identifiers. These tag names can be Common Data Elements (CDEs), which have a predefined meaning or semantics. By using this specification in a laboratory setting with increasing demands for digital pathology integration, we found that the data structure lacked the ability to cope with digital slide imaging in respect to web-enabled digital pathology systems and advanced scoring techniques. Materials and Methods: By employing user centric design, and observing behavior in relation to TMA scoring and associated data, the TMA DES format was extended to accommodate the current limitations. This was done with specific focus on developing a generic tool for handling any given scoring system, and utilizing data for multiple observations and observers. Results: DTDs were created to validate the extensions of the TMA DES protocol, and a test set of data containing scores for 6,708 TMA core images was generated. The XML was then read into an image processing algorithm to utilize the digital pathology data extensions, and scoring results were easily stored alongside the existing multiple pathologist scores. Conclusions: By extending the TMA DES format to include digital pathology data and customizable scoring systems for TMAs, the new system facilitates the collaboration between pathologists and organizations, and can be used in automatic or manual data analysis. This allows complying systems to effectively communicate complex and varied scoring data.