Patient and healthcare professionals' perceptions of a combined blood and faecal immunochemical test for excluding colorectal cancer diagnosis in primary care.

OBJECTIVES: To explore the perceptions of patients and healthcare professionals on Raman-faecal immunochemical test (FIT) as an alternative test for colorectal cancer exclusion in primary care. DESIGN: Semi-structured interviews within a feasibility study. SETTING: Patients presenting to primary care with colorectal symptoms and healthcare professionals working in primary and secondary care. PARTICIPANTS: A total of 23 patients and 12 healthcare professionals. METHODS: Patient participants were asked to complete a novel combined Raman-FIT test before being seen in secondary care. This study sought their opinions about the test. We also sought the views of healthcare professionals. FINDINGS: Patients and healthcare professionals agreed that Raman-FIT was a suitable test to be given in primary care. It aligned with routine practice and was a simple test for most patients to complete. CONCLUSIONS: Patients are willing and able to complete the Raman-FIT test in primary care. Raman-FIT may accelerate access to diagnosis with the potential to improve cancer outcomes. PATIENT AND PUBLIC INVOLVEMENT: Lay members (J. H. and I. H.) with experience and knowledge of colorectal cancer and screening contributed to developing, undertaking, and disseminating all aspects of the research. They were supported to collaborate as equal members of the research team. They were involved in developing the study as coapplicants, using personal experience to ensure that the research and its methods were relevant to the patient and public needs. Both prepared participant information sheets, coanalysed data, and contributed to study reporting and dissemination through papers, conference presentations and a lay summary.

Multi-cancer early detection test in symptomatic patients referred for cancer investigation in England and Wales (SYMPLIFY): a large-scale, observational cohort study.

BACKGROUND: Analysis of circulating tumour DNA could stratify cancer risk in symptomatic patients. We aimed to evaluate the performance of a methylation-based multicancer early detection (MCED) diagnostic test in symptomatic patients referred from primary care. METHODS: We did a multicentre, prospective, observational study at National Health Service (NHS) hospital sites in England and Wales. Participants aged 18 or older referred with non-specific symptoms or symptoms potentially due to gynaecological, lung, or upper or lower gastrointestinal cancers were included and gave a blood sample when they attended for urgent investigation. Participants were excluded if they had a history of or had received treatment for an invasive or haematological malignancy diagnosed within the preceding 3 years, were taking cytotoxic or demethylating agents that might interfere with the test, or had participated in another study of a GRAIL MCED test. Patients were followed until diagnostic resolution or up to 9 months. Cell-free DNA was isolated and the MCED test performed blinded to the clinical outcome. MCED predictions were compared with the diagnosis obtained by standard care to establish the primary outcomes of overall positive and negative predictive value, sensitivity, and specificity. Outcomes were assessed in participants with a valid MCED test result and diagnostic resolution. SYMPLIFY is registered with ISRCTN (ISRCTN10226380) and has completed follow-up at all sites. FINDINGS: 6238 participants were recruited between July 7 and Nov 30, 2021, across 44 hospital sites. 387 were excluded due to staff being unable to draw blood, sample errors, participant withdrawal, or identification of ineligibility after enrolment. Of 5851 clinically evaluable participants, 376 had no MCED test result and 14 had no information as to final diagnosis, resulting in 5461 included in the final cohort for analysis with an evaluable MCED test result and diagnostic outcome (368 [6·7%] with a cancer diagnosis and 5093 [93·3%] without a cancer diagnosis). The median age of participants was 61·9 years (IQR 53·4-73·0), 3609 (66·1%) were female and 1852 (33·9%) were male. The MCED test detected a cancer signal in 323 cases, in whom 244 cancer was diagnosed, yielding a positive predictive value of 75·5% (95% CI 70·5-80·1), negative predictive value of 97·6% (97·1-98·0), sensitivity of 66·3% (61·2-71·1), and specificity of 98·4% (98·1-98·8). Sensitivity increased with increasing age and cancer stage, from 24·2% (95% CI 16·0-34·1) in stage I to 95·3% (88·5-98·7) in stage IV. For cases in which a cancer signal was detected among patients with cancer, the MCED test's prediction of the site of origin was accurate in 85·2% (95% CI 79·8-89·3) of cases. Sensitivity 80·4% (95% CI 66·1-90·6) and negative predictive value 99·1% (98·2-99·6) were highest for patients with symptoms mandating investigation for upper gastrointestinal cancer. INTERPRETATION: This first large-scale prospective evaluation of an MCED diagnostic test in a symptomatic population demonstrates the feasibility of using an MCED test to assist clinicians with decisions regarding urgency and route of referral from primary care. Our data provide the basis for a prospective, interventional study in patients presenting to primary care with non-specific signs and symptoms. FUNDING: GRAIL Bio UK.

A novel blood based triage test for colorectal cancer in primary care: a pilot study.

BACKGROUND: The majority of colorectal cancers (CRCs) are detected after symptomatic presentation to primary care. Given the shared symptoms of CRC and benign disorders, it is challenging to manage the risk of missed diagnosis. Colonoscopy resources cannot keep pace with increasing demand. There is a pressing need for access to simple triage tools in primary care to help prioritise patients for referral. AIM: To evaluate the performance of a novel spectroscopy-based CRC blood test in primary care. DESIGN & SETTING: Mixed-methods pilot study of test performance and GP focus group discussions in South Wales. METHOD: Patients on the urgent suspected cancer (USC) pathway were recruited for the Raman spectroscopy (RS) test coupled to machine learning classification ('Raman-CRC') to identify CRC within the referred population. Qualitative focus group work evaluated the acceptability of the test in primary care by thematic analysis of focus group theorising. RESULTS: A total of 532 patients aged ≥50 years referred on the USC pathway were recruited from 27 GP practices. Twenty-nine patients (5.0%) were diagnosed with CRC. Raman-CRC identified CRC with sensitivity 95.7%, specificity 69.3% with area under curve (AUC) of 0.80 compared with colonoscopy as the reference test (248 patients). Stage I and II cancers were detected with 78.6% sensitivity. Focus group themes underlined the convenience of a blood test for the patient and the test's value as a risk assessment tool in primary care. CONCLUSION: The findings support this novel, non-invasive, blood-based method to prioritise those patients most likely to have CRC. Raman-CRC may accelerate access to diagnosis with potential to improve cancer outcomes.

Liquid Biopsy for Pancreatic Cancer Detection Using Infrared Spectroscopy.

Pancreatic cancer claims over 460,000 victims per year. The carbohydrate antigen (CA) 19-9 test is the blood test used for pancreatic cancer's detection; however, its levels can be raised in symptomatic patients with other non-malignant diseases, or with other tumors in the surrounding area. Attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy has demonstrated exceptional potential in cancer diagnostics, and its clinical implementation could represent a significant step towards early detection. This proof-of-concept study, investigating the use of ATR-FTIR spectroscopy on dried blood serum, focused on the discrimination of both cancer versus healthy control samples, and cancer versus symptomatic non-malignant control samples, as a novel liquid biopsy approach for pancreatic cancer diagnosis. Machine learning algorithms were applied, achieving results of up to 92% sensitivity and 88% specificity when discriminating between cancers (n = 100) and healthy controls (n = 100). An area under the curve (AUC) of 0.95 was obtained through receiver operating characteristic (ROC) analysis. Balanced sensitivity and specificity over 75%, with an AUC of 0.83, were achieved with cancers (n = 35) versus symptomatic controls (n = 35). Herein, we present these results as demonstration that our liquid biopsy approach could become a simple, minimally invasive, and reliable diagnostic test for pancreatic cancer detection.

Optimised Pre-Processing of Raman Spectra for Colorectal Cancer Detection Using High-Performance Computing.

Spectral pre-processing is an essential step in data analysis for biomedical diagnostic applications of Raman spectroscopy, allowing the removal of undesirable spectral contributions that could mask biological information used for diagnosis. However, due to the specificity of pre-processing for a given sample type and the vast number of potential pre-processing combinations, optimisation of pre-processing via a manual "trial and error" format is often time intensive with no guarantee that the chosen method is optimal for the sample type. Here we present the use of high-performance computing (HPC) to trial over 2.4 million pre-processing permutations to demonstrate the optimisation on the pre-processing of human serum Raman spectra for colorectal cancer detection. The effect of varying pre-processing order, using extended multiplicative scatter correction, spectral smoothing, baseline correction, binning and normalization was considered. Permutations were assessed on their ability to detect patients with disease using a random forest (RF) algorithm trained with 102 patients (510 spectra) and independently tested with a set of 439 patients (1317 spectra) in a primary care patient cohort. Optimising via HPC enables improved performance in diagnostic abilities, with sensitivity increasing by 14.6%, specificity increasing by 6.9%, positive predictive value increasing by 3.4%, and negative predictive value increasing by 2.4% when compared to a standard pre-processing optimisation. Ultimate values of these metrics are very important for diagnostic adoption, and once diagnostics demonstrate good accuracy these types of optimisations can make a significant difference to roll-out of a test and demonstrating advantages over existing tests. We also provide tips/recommendations for pre-processing optimisation without the use of HPC. From the HPC permutations, recommendations for appropriate parameter constraints for conducting a more basic pre-processing optimisation are also detailed, thus helping model development for researchers not having access to HPC.

Rapid analysis of disease state in liquid human serum combining infrared spectroscopy and "digital drying".

In recent years, the diagnosis of brain tumors has been investigated with attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy on dried human serum samples to eliminate spectral interferences of the water component, with promising results. This research evaluates ATR-FTIR on both liquid and air-dried samples to investigate "digital drying" as an alternative approach for the analysis of spectra obtained from liquid samples. Digital drying approaches, consisting of water subtraction and least-squares method, have demonstrated a greater random forest (RF) classification performance than the air-dried spectra approach when discriminating cancer vs control samples, reaching sensitivity values higher than 93.0% and specificity values higher than 83.0%. Moreover, quantum cascade laser infrared (QCL-IR) based spectroscopic imaging is utilized on liquid samples to assess the implications of a deep-penetration light source on disease classification. The RF classification of QCL-IR data has provided sensitivity and specificity amounting to 85.1% and 75.3% respectively.

A high-throughput serum Raman spectroscopy platform and methodology for colorectal cancer diagnostics.

Vibrational spectroscopic techniques such as Raman spectroscopy and Fourier transform infrared spectroscopy (FTIR) have huge potential for the analysis of biological specimens. The techniques allow the user to gain label-free, non-destructive biochemical information about a given sample. Previous studies using vibrational spectroscopy with the specific application of diagnosing colorectal diseases such as cancer have mainly focused on in vivo or in vitro studies of tissue specimens using microscopy or probe based techniques. There have been few studies of vibrational spectroscopic techniques based on the analysis of blood serum for the advancement of colorectal cancer diagnostics. With growing interest in the field of liquid biopsies, this study presents the development of a high-throughput (HT) serum Raman spectroscopy platform and methodology and compares dry and liquid data acquisition of serum samples. This work considers factors contributing to translatability of the methodologies such as HT design, inter-user variability and sample handling effects on diagnostic capability. The HT Raman methods were tested on a pilot dataset of serum from 30 cancer patients and 30 matched control patients using statistical analysis via cross-validated PLS-DA with a maximum achieved a sensitivity of 83% and specificity of 83% for detecting colorectal cancer.

DNA hypermethylation as a predictor of extramural vascular invasion (EMVI) in rectal cancer.

PURPOSE: DNA hypermethylation in gene promoter regions (CpG islands) is emerging as an important pathway in colorectal cancer tumourigenesis. Whilst genetic mutations have been associated with extramural vascular invasion (EMVI) in rectal cancer, no such association has yet been made with epigenetic factors. METHODS: 100 consecutive neoadjuvant-naïve patients undergoing curative surgery for rectal were classified according to the presence or absence of EMVI on histopathological examination. DNA was extracted from tumours and subjected to bisulfite conversion and methylation-specific PCR to determine CIMP status (high, intermediate, or low; according to a validated panel of 8 genes). CIMP status was correlated with EMVI status, histopathological, clinical, and demographic variables, in addition to overall (OS) and disease free (DFS) survival. RESULTS: 51 patients were characterised as CIMP-low, 48 CIMP-intermediate, and one patient CIMP-high. EMVI-positivity was associated with CIMP-intermediate epigenotype (p < 0.001). Patients with EMVI-positive tumours were found to have significantly more advanced disease by pT, pN, and pAJCC categorisation (p = 0.002, p < 0.001, and = p < 0.001, respectively). EMVI-positivity was significantly associated with the requirement for adjuvant chemotherapy (p < 0.001), and worse DFS but not OS (p = 0.012 and p = 0.052). CONCLUSIONS: Given the association between CIMP-intermediate epigenotype and EMVI-positivity, and the subsequent disadvantage in pathological stage, requirement for adjuvant therapy and worse survival, tumour epigenotyping could potentially play an important role in personalising patients' cancer care. Further work is required to understand the mechanisms that underlie the observed effect, with the hope that they may provide novel opportunities for intervention and inform treatment decisions in rectal cancer.

Protocol for a multicentre randomised feasibility trial evaluating early Surgery Alone In LOw Rectal cancer (SAILOR).

INTRODUCTION: There are 11 500 rectal cancers diagnosed annually in the UK. Although surgery remains the primary treatment, there is evidence that preoperative radiotherapy (RT) improves local recurrence rates. High-quality surgery in rectal cancer is equally important in minimising local recurrence. Advances in MRI-guided prediction of resection margin status and improvements in abdominoperineal excision of the rectum (APER) technique supports a reassessment of the contribution of preoperative RT. A more selective approach to RT may be appropriate given the associated toxicity. METHODS AND ANALYSIS: This trial will explore the feasibility of a definitive trial evaluating the omission of RT in resectable low rectal cancer requiring APER. It will test the feasibility of randomising patients to (1) standard care (neoadjuvant long course RT±chemotherapy and APER, or (2) APER surgery alone for cT2/T3ab N0/1 low rectal cancer with clear predicted resection margins on MRI. RT schedule will be 45 Gy over 5 weeks as current standard, with restaging and surgery after 8-12 weeks. Recruitment will be for 24 months with a minimum 12-month follow-up. OBJECTIVES: Objectives include testing the ability to recruit, consent and retain patients, to quantify the number of patients eligible for a definitive trial and to test feasibility of outcomes measures. These include locoregional recurrence rates, distance to circumferential resection margin, toxicity and surgical complications including perineal wound healing, quality of life and economic analysis. The quality of MRI staging, RT delivery and surgical specimen quality will be closely monitored. ETHICS AND DISSEMINATION: The trial is approved by the Regional Ethics Committee and Health Research Authority (HRA) or equivalent. Written informed consent will be obtained. Serious adverse events will be reported to Swansea Trials Unit (STU), the ethics committee and trial sites. Trial results will be submitted for peer review publication and to trial participants. TRIAL REGISTRATION NUMBER: ISRCTN02406823.

Role of Raman spectroscopy and surface enhanced Raman spectroscopy in colorectal cancer.

Colorectal cancer (CRC) is the fourth most common cancer in the United Kingdom and is the second largest cause of cancer related death in the United Kingdom after lung cancer. Currently in the United Kingdom there is not a diagnostic test that has sufficient differentiation between patients with cancer and those without cancer so the current referral system relies on symptomatic presentation in a primary care setting. Raman spectroscopy and surface enhanced Raman spectroscopy (SERS) are forms of vibrational spectroscopy that offer a non-destructive method to gain molecular information about biological samples. The techniques offer a wide range of applications from in vivo or in vitro diagnostics using endoscopic probes, to the use of micro-spectrometers for analysis of biofluids. The techniques have the potential to detect molecular changes prior to any morphological changes occurring in the tissue and therefore could offer many possibilities to aid the detection of CRC. The purpose of this review is to look at the current state of diagnostic technology in the United Kingdom. The development of Raman spectroscopy and SERS in clinical applications relation for CRC will then be discussed. Finally, future areas of research of Raman/SERS as a clinical tool for the diagnosis of CRC are also discussed.

Review of the development of DNA methylation as a marker of response to neoadjuvant therapy and outcomes in rectal cancer.

There is much debate around the preoperative treatment of colorectal cancer and, in particular, neoadjuvant chemoradiotherapy in locally advanced rectal cancer. This treatment carries a significant risk of harmful side effects and has a highly variable response rate. Predictive biomarkers have been the subject of a great deal of study with the aim of pretreatment risk stratification in order to more accurately determine which patients will derive the most benefit and least harm from these treatments. The study of epigenetics in colorectal cancer is relatively recent, and distinct patterns of aberrant DNA methylation, in particular the cytosine-phosphate-guanine (CpG) island methylator phenotype (CIMP), have been demonstrated in colorectal cancer, and their characterisation and significance are under debate, particularly in rectal cancer. These patterns of DNA methylation have been associated with differences in response to therapy and treatment outcomes and therefore have the potential to be used as biomarkers in tailored therapy regimes for patients with rectal cancer. This review aims to summarise the current state of the art in rectal cancer, with particular regard to the determination of DNA methylation patterns, the CpG island methylator phenotype and its potential as a novel biomarker in rectal cancer treatment and prediction of outcomes and response after neoadjuvant chemoradiotherapy.

Viral IL-6 blocks neutrophil infiltration during acute inflammation.

Pathologies arising as a consequence of human herpesvirus-8 (HHV8) infections are closely associated with the autocrine activity of a HHV8 encoded IL-6 (vIL-6), which promotes proliferation of infected cells and their resistance to apoptosis. In this present report, studies show that vIL-6 may also be important in influencing the host's immunological response to secondary infections. Using peritoneal inflammation as a model of acute bacterial infection, vIL-6 was found to specifically block neutrophil recruitment in vivo through regulation of inflammatory chemokine expression. This response was substantiated in vitro where activation of STAT3 in human peritoneal mesothelial cells by vIL-6 was associated with enhanced CCL2 release. Although vIL-6 did not effect CXCL8 production, IL-1beta-induced secretion of this neutrophil-activating chemokine was significantly suppressed by vIL-6. These data suggest that vIL-6 has the capacity to suppress innate immune responses and thereby influence the outcome of opportunistic infections in HHV8-associated disease.

Differential regulation of neutrophil-activating chemokines by IL-6 and its soluble receptor isoforms.

Interleukin-6 signaling via its soluble receptor (sIL-6R) differentially regulates inflammatory chemokine expression and leukocyte apoptosis to coordinate transition from neutrophil to mononuclear cell infiltration. sIL-6R activities may, however, be influenced in vivo by the occurrence of two sIL-6R isoforms that are released as a consequence of differential mRNA splicing (DS) or proteolytic cleavage (PC) of the cognate IL-6R (termed DS- and PC-sIL-6R). Using human peritoneal mesothelial cells and a murine model of peritoneal inflammation, studies described in this work have compared the ability of both isoforms to regulate neutrophil recruitment. In this respect, DS- and PC-sIL-6R were comparable in their activities; however, these studies emphasized that IL-6 trans signaling differentially controls neutrophil-activating CXC chemokine expression. In vitro, stimulation of mesothelial cells with IL-6 in combination with either DS-sIL-6R or PC-sIL-6R showed no induction of CXC chemokine ligand (CXCL)1 (GRO alpha) and CXCL8 (IL-8), whereas both isoforms enhanced CXCL5 (ENA-78) and CXCL6 (granulocyte chemotactic protein-2) expression. Moreover, when complexed with IL-6, both isoforms specifically inhibited the IL-1 beta-induced secretion of CXCL8. These findings were paralleled in vivo, in which induction of peritoneal inflammation in IL-6-deficient (IL-6(-/-)) mice resulted in enhanced keratinocyte-derived chemokine and macrophage-inflammatory protein-2 (the murine equivalent of CXCL1 and CXCL8) levels, but reduced LPS-induced CXC chemokine (the murine equivalent of CXCL5) expression. Reconstitution of IL-6 signaling in IL-6(-/-) mice with IL-6 and its soluble receptor isoforms corrected this chemokine imbalance and suppressed overall neutrophil infiltration. These data confirm that sIL-6R-mediated signaling primarily limits neutrophil influx; however, induction of CXCL5 and CXCL6 may regulate other neutrophil responses.

Diagnosis and management of primary aldosteronism.

Identifying primary aldosteronism within the hypertensive population is an important clinical challenge, as most patients with a unilateral source of excess aldosterone secretion are amenable to surgical cure. At least 20% of patients with primary aldosteronism have normal serum potassium levels. Therefore, screening tests should not be based on recognition of hypokalemia alone. Rather, the diagnosis should depend on identifying renin suppression and measuring the ratio of plasma aldosterone concentration to plasma renin activity. The diagnosis may be confirmed by performing an aldosterone suppression test after oral salt loading. Once primary aldosteronism has been established, it is necessary to exclude glucocorticoid-remediable aldosteronism and then proceed to localization studies. Detecting a unilateral source of aldosterone, usually due to an adenoma (Conn syndrome), is achieved by postural hormonal testing and confirmed by selective venous sampling (SVS) with measurement of aldosterone concentrations (expressed as the aldosterone/cortisol ratio) in each adrenal vein. SVS is enjoying a revival in many institutions as it is more sensitive and specific than either cross-sectional imaging or scintigraphy and has the potential to influence significantly both the diagnosis and clinical decision-making. Patients with unilateral disease are ideally treated by laparoscopic adrenalectomy. Patients in whom localization is not achieved usually have bilateral adrenal hyperplasia and are treated medically.