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The increasing prevalence of diabetes and stroke is a major global public health concern. Specifically, acute stroke patients, with pre-existing diabetes, pose a clinical challenge. It is established that diabetes is associated with a worse prognosis after acute stroke and the various biological factors that mediate poor recovery profiles in diabetic patients is unknown. The level of association and impact of diabetes, in the setting of reperfusion therapy, is yet to be determined. This article presents a comprehensive overview of the current knowledge of the role of diabetes in stroke, therapeutic strategies for primary and secondary prevention of cardiovascular disease and/or stroke in diabetes, and various therapeutic considerations that may apply during pre-stroke, acute, sub-acute and post-stroke stages. The early diagnosis of diabetes as a comorbidity for stroke, as well as tailored post-stroke management of diabetes, is pivotal to our efforts to limit the burden. Increasing awareness and involvement of neurologists in the management of diabetes and other cardiovascular risk factors is desirable towards improving stroke prevention and efficacy of reperfusion therapy in acute stroke patients with diabetes.

There is an ongoing need for accurate prognostic biomarkers in the milieu of acute ischemic stroke (AIS) receiving reperfusion therapy. Neutrophil-lymphocyte ratio (NLR) has been implicated in emergency medicine and acute stroke setting as an important biomarker in the prognosis of patients. However, there are ongoing questions around its accuracy and translation into clinical practice given suboptimal sensitivity and specificity results, as well as varying thresholds and lack of clarity around which NLR time points are most clinically indicative. This article provides a comprehensive overview of the role of NLR in AIS patients receiving reperfusion therapy and perspectives on areas of future research. NLR may be an important biomarker in risk stratifying patients in AIS to identify and select those who are more likely to benefit from reperfusion therapy. Appropriate clinical decision-making tools and models are required to harness the predictive value of NLR, which could be useful in identifying and monitoring high-risk patients to guide early treatment and achieve improved outcomes. Our understanding of the role of NLR in the immunopathogenesis of AIS is also suboptimal, which hinders the ability to translate this into clinical practice.

For the past two decades, microsatellite instability (MSI) has been reported as a robust clinical biomarker associated with survival advantage attributed to its immunogenicity. However, MSI is also associated with high-risk adverse pathological features (poorly differentiated, mucinous, signet cell, higher grade) and exhibits a double-edged sword phenomenon. We performed a systematic review and meta-analysis to evaluate the rate of dissemination and the prognosis of early and advanced stage colorectal cancer based on MSI status. A systematic literature search of original studies was performed on Ovid searching MEDLINE, Embase, Cochrane Database of Systematic Reviews, American College of Physicians ACP Journal Club, Database of Abstracts of Reviews of Effects DARE, Clinical Trials databases from inception of database to June 2019. Colorectal cancer, microsatellite instability, genomic instability and DNA mismatch repair were used as key words or MeSH terms. The Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) reporting guideline was followed. Data were pooled using a random-effects model with odds ratio (OR) as the effect size. Statistical analysis was performed using RevMan ver 5.3 Cochrane Collaboration. From 5288 studies, 136 met the inclusion criteria (n = 92,035; MSI-H 11,746 (13%)). Overall, MSI-H was associated with improved OS (OR, 0.81; 95% CI 0.73–0.90), DFS (OR, 0.73; 95% CI 0.66–0.81) and DSS (OR, 0.69; 95% CI 0.52–0.90). Importantly, MSI-H had a protective effect against dissemination with a significantly lower rate of lymph node and distant metastases. By stage, the protective effect of MSI-H in terms of OS and DFS was observed clearly in stage II and stage III. Survival in stage I CRC was excellent irrespective of MSI status. In stage IV CRC, without immunotherapy, MSI-H was not associated with any survival benefit. MSI-H CRC was associated with an overall survival benefit with a lower rate of dissemination. Survival benefit was clearly evident in both stage II and III CRC, but MSI-H was neither a robust prognostic marker in stage I nor stage IV CRC without immunotherapy.

Background Recent studies in non-colorectal malignancy have associated T resident memory (TRM) cells with improved patient survival. It is unknown if TRM plays a role in colorectal cancer (CRC). Aim To examine the potential role of TRM cells in providing immunogenicity in CRC stratified by microsatellite instability (MSI) and BRAF status. Methods Patients with known MSI and BRAF mutation status were eligible for inclusion in this study. CRC tumour sections stained with haematoxylin and eosin were microscopically reviewed and the images scanned prior to assessment for location of invading edge and core of tumour. Sequential sections were prepared for quantitative multiplex immunohistochemistry (IHC) staining. Opal Multiplex IHC staining was performed with appropriate positive and negative controls and imaged using a standard fluorescent microscope fitted with a spectral scanning camera (Mantra) in conjunction with Mantra snap software. Images were unmixed and annotated in inForm 2.2.0. Statistical analysis was performed using Graphpad Prism Version 7 and Stata Version 15. Results Seventy-two patients with known MSI and BRAF status were included in the study. All patients were assessed for MSI by IHC and high resolution capillary electrophoresis testing and 44 of these patients successfully underwent quantitative multiplex IHC staining. Overall, there was a statistically significant increase in CD8+ TRM cells in the MSI (BRAF mutant and wild type) group over the microsatellite stable (MSS) group. There was a statistically significant difference in CD8+ TRM between high level MSI (MSI-H):BRAF mutant [22.57, 95% confidence interval (CI): 14.31-30.84] vs MSS [8.031 (95%CI: 4.698-11.36)], P = 0.0076 andMSI-H:BRAF wild type [16.18 (95%CI: 10.44-21.93)] vs MSS [8.031 (95%CI: 4.698-11.36)], P = 0.0279. There was no statistically significant difference in CD8 T cells (both CD8+CD103- and CD8+CD103+TRM) between MSI-H: BRAF mutant and wild type CRC. Conclusion This study has shown that CD8+ TRM are found in greater abundance in MSI-H CRC, both BRAF mutant and MSI-H:BRAF wild type, when compared with their MSS counterpart. CD8+ TRM may play a role in the immunogenicity in MSI-H CRC (BRAF mutant and BRAF wild type). Further studies should focus on the potential immunogenic qualities of TRM cells and investigate potential immunotherapeutic approaches to improve treatment and survival associated with CRC.

Background There is significant variation in attitude both towards the role of microsatellite instability (MSI) in predicting prognosis, and towards its role in guiding which Stage II colon cancer patients may benefit from adjuvant chemotherapy. Aim To examine the current status of specialist attitudes towards MSI in guiding prognosis and adjuvant therapy in stage II colon cancer. Methods The Pathology in Colon Cancer, Prognosis and Uptake of Adjuvant Therapy (PiCC UP) Australia and New Zealand questionnaire was distributed to colorectal surgeons, medical oncologists and pathologists after institutional board approval. A 5-scale Likert score was used to assess attitudes towards 23 pathological features for prognosis and 18 features for adjuvant therapy. Data were analysed using a rating scale and graded response model in item response theory (IRT) on STATA (Stata MP, version 15; StataCorp LP). Results 164 specialists (45 oncologists, 86 surgeons and 33 pathologists) participated. 80.5% regularly attended colorectal multidisciplinary team (MDT) meetings. 89.63% and 59.26% of specialists reported that MSI status was likely or definitely to influence prognosis in colon cancer and recommendations for adjuvant therapy in Stage II colon cancer respectively. IRT modelling was achieved in 17 pathological features for prognosis. MSI IRT score was 4.47 (95% CI: 4.05-4.68). IRT modelling was achieved in 10 pathological features for adjuvant therapy. MSI IRT score was 3.62 (2.89-4.15). MSI ranked 10 (of 17) in order of importance in determining prognosis and ranked three (of 10) in guiding adjuvant therapy. Conclusion MSI status is considered an important biomarker when selecting patients for adjuvant therapy in Stage II colon cancer. MSI is also considered useful in prognostication of colon cancer. MSI status was ranked similar to the tumour grade of differentiation and the presence of perineural invasion.

Microsatellite instability (MSI) in colorectal cancer (CRC) is a marker of immunogenicity and is associated with an increased abundance of tumour infiltrating lymphocytes (TILs). In this subgroup of colorectal cancer, it is unknown if these characteristics translate into a measurable difference in circulating tumour cell (CTC) release into peripheral circulation. This is the first study to compare MSI status with the prevalence of circulating CTCs in the peri-operative colorectal surgery setting. For this purpose, 20 patients who underwent CRC surgery with curative intent were enrolled in the study, and peripheral venous blood was collected at pre- (t1), intra- (t2), immediately post-operative (t3), and 14&ndash, 16 h post-operative (t4) time points. Of these, one patient was excluded due to insufficient blood sample. CTCs were isolated from 19 patients using the IsofluxTM system, and the data were analysed using the STATA statistical package. CTC number was presented as the mean values, and comparisons were made using the Student t-test. There was a trend toward increased CTC presence in the MSI-high (H) CRC group, but this was not statistically significant. In addition, a Poisson regression was performed adjusting for stage (I-IV). This demonstrated no significant difference between the two MSI groups for pre-operative time point t1. However, time points t2, t3, and t4 were associated with increased CTC presence for MSI-H CRCs. In conclusion, there was a trend toward increased CTC release pre-, intra-, and post-operatively in MSI-H CRCs, but this was only statistically significant intra-operatively. When adjusting for stage, MSI-H was associated with an increase in CTC numbers intra-operatively and post-operatively, but not pre-operatively.

In vitro models of the liver have led to important insights into the pathogenesis of liver disease. These models are essential tools in the discovery and preclinical stages of drug development. The clinical application of these models is also emerging as a promising avenue for validating genetic target-matched treatments, in a precision medicine approach to treatment. Recent advances in ‘liver-on-a-chip’ technology and liver organoid research have opened up new opportunities for the functional and clinical use of organotypic in vitro models. This chapter focuses on the currently available in vitro liver models and the opportunities and limitations they present in the context of evaluating their use in disease modeling, drug discovery, and clinical application.

Aims: There is limited data on health-related quality of life (HRQoL) in locally advanced rectal cancer. We assessed HRQoL before, during and after neoadjuvant chemoradiation, correlated this to corresponding clinician-reported adverse events (CR-AEs) and explored disparities between patients of Asian ethnicity versus Caucasians. Correlation between HRQoL and treatment response was also assessed. Methods: A consecutive sample of patients was recruited. HRQoL was assessed with the EORTC QLQ-C30 before chemoradiation, week three of chemoradiation and one-week pre-surgery. Clinical variables including CR-AEs were recorded at these time-points. Patients self-reported socio-demographic variables. Treatment response was assessed by the tumour regression grade. HRQoL data were analysed with multilevel models. Results: Fifty-one patients were recruited. HRQoL completion rates were &ge, 86%. Cognitive and role functioning worsened significantly during treatment. Emotional, role and social functioning improved significantly at pre-surgery. Fatigue and nausea/vomiting worsened during treatment while fatigue, appetite loss, diarrhoea and financial difficulties improved from treatment to pre-surgery. Almost 30% of the cohort were Asian ethnicity. Differences were found in multiple HRQoL domains between Asians and Caucasians, with Asians faring worse. Significant differences were evident in physical, role and cognitive functioning, and in seven out of the 8 symptom scales. The correlation between patient-reported outcomes and clinician-reported outcomes was weak, with diarrhoea having the strongest correlation (r = 0.58). Vomiting during treatment correlated with poor response, whilst baseline constipation correlated with good response. Conclusion: Chemoradiation for locally advanced rectal cancer affects multiple HRQoL domains. Our findings highlight the importance of psychological aspects of treatment. Significant differences were identified between the Asian and Caucasian populations, with Asians consistently performing worse. Poor correlations between patient and clinician reporting strongly support the inclusion of patient-reported outcomes in clinical studies. HRQoL domains of vomiting and constipation are potential biomarkers of treatment response.

Although durable clinical responses are achieved in a significant number of patients given Immune checkpoint inhibitors (ICI), like anti-CTLA-4 and anti-PD-1 inhibitors, some of the cancers have shown little or no response to ICI therapy. Even within the known responsive cancers, there is often a subset of non-responsive patients. Due to the accelerated FDA approval of these immunotherapies, the biomarker development has not been able to keep pace. Appropriate predictive, prognostic and surrogate biomarkers are needed to maximally exploit the benefits from ICI therapy for correct and timely stratification of patients to treatment, for monitoring treatment effect, and for avoiding costs and unwanted toxicities when therapy is likely to be ineffective. As the number of clinical trials exploring the utility of these treatments, both as stand-alone and as combination therapy for several cancers is escalating dramatically, the need for appropriate biomarkers is further amplified. This review discusses the potential biomarkers being investigated in ICI therapies, focusing mainly on immunohistochemical expression of PDL-1 and the immune correlates. Various immune components discussed here include the cells of innate (natural killer or NK cells) and adaptive (CD4+ and CD8+ cells) immunity, regulatory and inhibitory immune cells (regulatory T cells or Tregs and myeloid derived suppressor cells or MDSCs), as well as cytokines. Immune checkpoint molecule, programmed death receptor ligand-1 (PD-L1) and various molecules and pathways influencing its expression are also discussed.

Colorectal cancers (CRCs) have been identified as potential targets for immunotherapy with programmed cell death (PD)-1 inhibitors. English-language publications from MedLine and Embase that evaluated PD-1/PD ligand 1 (PD-L1) in the CRC tumor microenvironment and clinical trials that assessed PD-1 inhibitors were included. Sixteen abstracts were screened. Fifteen met the inclusion criteria. After review of the full texts, this resulted in a final reference list of 8 studies eligible for review. Five studies that assessed PD-1/PD-L1 in CRC and 3 trials that assessed PD-1 inhibitors were included. PD–1-positive (PD-1 + ) tumor-infiltrating lymphocytes and PD-L1 + cancer cells featured more prominently in high-level microsatellite instability (MSI-H) CRCs compared with microsatellite stable (MSS) CRCs, except in 1 study in which PD-L1 expression was higher in MSS CRCs. In the 3 trials that assessed PD-1 inhibitor, all 3 studies recruited patients with metastatic CRC (mCRC). One study also included patients with recurrent CRC. The objective response according to the Response Evaluation Criteria in Solid Tumors criteria was 0% (19 CRC patients with unknown microsatellite instability status) in the nivolumab study. In the pembrolizumab study, the objective response to PD-1 inhibitor was 40% and 0% in patients with MSI-H and MSS mCRC, respectively (10 patients in the MSI-H group, 18 patients in the MSS group). Seventy-eight percent of the patients in the MSI-H mCRC group compared with 11% in the MSS mCRC group ( P

There are concerns that a substantial proportion of published research data is not reproducible, which may partially explain the frequent failure to translate pre-clinical results to clinical care. High-quality cancer biospecimens are needed for robust, reproducible research findings, with most researchers obtaining these specimens from cancer biobanks or tumour banks. This review provides an overview of the types of quality control (QC) activities conducted within cancer biobanks that pertain to biospecimen quality and of biospecimen quality reporting tools, including SPREC and BRISQ. We examine how QC assay results and other biospecimen data are communicated from biobanks to researchers, and whether these activities lead to improved biospecimen quality reporting within the literature and/or to improved research outcomes. We also discuss operational factors that limit QC activities within biobanks and evidence gaps requiring further research. In summary, whereas the provision of quality biospecimens is a common aim of cancer biobanks, QC activities remain underreported and are rarely discussed in the literature, compared with other aspects of biobank operations. Further research is required to determine how biobanks can most efficiently optimise biospecimen quality, and how communication between biobanks and researchers can be improved.

In 2011, Watson and Barnes proposed a schema for classifying biobanks into 3 groups (mono-, oligo-, and poly-user), primarily based upon biospecimen access policies. We used results from a recent comprehensive survey of cancer biobanks in New South Wales, Australia to assess the applicability of this biobank classification schema in an Australian setting. Cancer biobanks were identified using publically available data, and by consulting with research managers. A comprehensive survey was developed and administered through a face-to-face setting. Data were analyzed using Microsoft Excel™ 2010 and IBM SPSS Statistics™ version 21.0. The cancer biobank cohort (n=23) represented 5 mono-user biobanks, 7 oligo-user biobanks, and 11 poly-user biobanks, and was analyzed as two groups (mono-/oligo- versus poly-user biobanks). Poly-user biobanks employed significantly more full-time equivalent staff, and were significantly more likely to have a website, share staff between biobanks, access governance support, utilize quality control measures, be aware of biobanking best practice documents, and offer staff training. Mono-/oligo-user biobanks were significantly more likely to seek advice from other biobanks. Our results further delineate a biobank classification system that is primarily based on access policy, and demonstrate its relevance in an Australian setting.

There is accumulating evidence for circulating tumour cells (CTCs) and circulating tumour nucleic acids (ctNAs) as prognostic and predictive biomarkers in colorectal cancer. Their role in the perioperative setting is evolving. These blood-borne biomarkers can potentially demonstrate tumour dissemination at time of colorectal cancer surgery and estimate the completeness of a surgical resection. CTCs and circulating ctNA levels at time of surgery, and persistent levels post-surgery, may correlate with poorer patient outcomes. These biomarkers can be utilised to refine surgical techniques to minimise tumour dissemination and determine the need for adjuvant therapy.

Introduction: 5'-Azacitidine (AZA), a DNA demethylating agent, is the primary drug for the treatment of high-risk Myelodysplastic Syndrome (MDS) and Chronic Myelomonocytic Leukaemia (CMML). Response is associated with improved survival. However, only half of patients respond, and these responses are rarely durable. We recently reported that primary AZA resistance is associated with a molecular signature of cell cycle quiescence within bone marrow (BM) hematopoietic progenitor cells (Unnikrishnan et al, Cell Reports, 20:572-585 (2017)). As DNA incorporation of the deoxyribonucleic form of AZA (5-aza-2′-deoxycytidine, DAC) occurs during DNA replication, cell cycle quiescence is predicted to lead to less DAC in DNA and concomitantly less DNA demethylation. We recently developed a quantitative multi-parameter assay, AZA-MS (Unnikrishnan, Vo et al, Leukemia 32:900-910 (2018)), to measure the intracellular dynamics of AZA in patients. Using AZA-MS, we reported data supporting the predicted resistance model. CC486 is an oral formulation of AZA. A 28-day cycle of CC486 involves 21 continuous days (21/28) versus the standard 7/28 subcutaneous (SC) injection AZA scheme. Whether levels of in vivo DAC incorporation into DNA during a cycle of CC486 are comparable with that of SC AZA is unknown. AZA-MS provides us with a unique opportunity to empirically assess the in vivo intracellular dynamics of SC versus oral AZA. Study Design and Methods: To directly assess in vivo DAC incorporation and concomitant DNA demethylation with SC AZA and CC486 in the same patient, we initiated a phase II clinical trial (NCT03493646; Fig A). MDS (IPSS; intermediate-2 or high-risk), CMML (bone marrow [BM] blasts 10-29%) and AML (20-30%) patients were recruited for six cycles of SC AZA (75mg/m^2/day for 7/28 days) followed by six cycles of CC486 (100mg bid for 21/28 days in C7-C8 and 150mg bid for 21/28 in C9-C12). Clinical response was assessed at the end of C6 and C12 using International Working Group criteria. Clinical responders and non-responders to SC AZA at C6 received CC486 from C7 onwards. From each patient, 36 peripheral blood (PB) samples and five BM samples were collected over the study period. DNA, RNA and intracellular fractions were isolated from the PB MNCs, for intracellular DAC/AZA measurements by AZA-MS (primary endpoint; Fig A). BM MNCs were utilised for AZA-MS as well as flow cytometry-based cell cycle measurements (secondary endpoint). Results: 31 of 42 consented patients have commenced treatment since trial opening (Fig B-C). We applied the AZA-MS assay on the longitudinal PB and BM samples collected from the seven patients who had completed six months AZA and commenced CC486 as at 26th June 2019 (Fig D). DAC incorporation into DNA and DNA methylation levels were quantified within the same cells, in addition to measuring other parameters (Fig E). As represented by patient 61213-005 (Fig F) who had a complete response (CR) at cycle 6, after 7 days of injection AZA we observed robust incorporation of DAC within PB MNCs (left panel, Fig F) together with concomitant DNA demethylation (right panel, Fig F). DAC levels diminished upon cessation of AZA within a cycle, with corresponding increases in DNA methylation. There were quantitatively higher levels of DAC incorporated in DNA during SC AZA cycles versus CC486. The trend observed is also appreciated from 2.3x higher area under the curve (AUC) measurements in 61213-005 during the SC AZA cycle. DAC incorporation was higher at C9/10 (CC486 150mg bid 21/28) than at C7/8 (CC486 100mg bid 21/28) without appreciable changes in DNA demethylation. During SC AZA cycles, higher DAC levels (top panel, Fig G) and greater DNA methylation (lower panel, Fig G) were seen in the BM MNCs. In a non-responding patient at cycle 6 (61290-002, SD), we saw less DAC incorporation and DNA demethylation (Fig H). We also observed a positive correlation between baseline proportions of cycling BM cells (LIN-CD34+CD38+) and the amount of DAC incorporated in BM MNCs at C1 day 8 (Fig I). Conclusion: AZA-MS can be used to reliably measure in vivo DAC incorporation and concomitant DNA demethylation in PB MNCs and inform appropriate CC486 dosing. Figure Disclosures Unnikrishnan: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Fong:Astellas: Consultancy; Novartis: Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; Amgen: Consultancy, Research Funding, Speakers Bureau. Roncolato:St. George Hospital: Employment. Enjeti:Roche: Honoraria, Speakers Bureau; Bayer and Sanofi: Honoraria, Speakers Bureau; Astellas: Consultancy; Novartis: Consultancy; Abbvie: Consultancy. Hertzberg:BMS: Membership on an entity's Board of Directors or advisory committees; F. Hoffmann-La Roche Ltd: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees. Polizzotto:Janssen: Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Research Funding; ViiV: Research Funding. Pimanda:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Background Colorectal cancers with microsatellite instability (MSI) are associated with abundance of tumour infiltrating lymphocytes (TILs). Recent studies have shown that a proportion of TILs may actually be resident memory T cells, not just a continuous circulation of T cells. Methods Patients with known MSI and BRAF status were eligible for inclusion in this study. Histopathology slides prepared with haematoxylin and eosin were reviewed using an Olympus BX53 microscope to examine the tumour invading edge and core. These slides were then scanned electronically and reexamined with both the investigator and a pathologist. Blocks from these representative slides were recut using a microtome in preparation for quantitative multiplex immunofluorescence staining. All immunofluorescence staining was carried out on 4-µm-thick sections using an Autostainer Plus (Dako – Agilent Technologies) with appropriate positive and negative controls. Opal Multiplex IHC Assay kit (PerkinElmer) was used. Statistical analysis was performed using Graphpad Prism Version 7 and Stata Version 15. Results 72 patients with known MSI and BRAF status were eligible for inclusion in this study. 36 of these patients were successfully underwent quantitative multiplex immunofluorescence staining. Overall, there was a statistically significant increase in T cells in the MSI BRAF mutant and wild type group over the MSS group. There was a statistically significant difference in Trm between MSI-H BRAF mutant vs. MSS (mean/mm2: 2.922 (2.4239-3.4201) vs. 0.9854 (0.8162-1.1546) p = 0.0002). There was also a statistically significant difference between MSI-H BRAF wt vs. MSS (mean/ mm2: 2.119 (1.7603-2.4777) vs. 0.9854 (0.8162-1.1546) p = 0.0002). The difference between MSI-H BRAF mutant and wild type was not statistically significant. Conclusions This study has shown that resident memory T cells are in greater abundance in MSI-H colorectal cancers compared to their MSS counterpart. Trms may play a role in the immunogenicity of MSI-H colorectal cancers, and may be a target for immune-related therapy. Legal entity responsible for the study The authors. Funding Has not received any funding. Disclosure All authors have declared no conflicts of interest.

Background Microsatellite instability (MSI) is thought to be a marker of immunogenicity and better prognosis in colorectal cancer (CRC). However, the mechanism by which MSI confers a survival advantage is not well known and there is a range of results reported in the literature. Methods A systematic literature search of original studies was performed on Ovid searching Medline, Embase, Cochrane Library, CINAHL, Clinical Trials databases from inception of database to current. Data extracted included age, stage, MSI-H, MSS and MSI-L, proximal (right) vs. distal (left), colon vs. rectal, BRAF status, type of MSI or IHC test used, incidence of Lynch within cohort. The primary endpoint was survival (overall survival (OS), disease/relapse free survival (DFS) and disease (cancer) specific survival DSS). Statistical analysis was performed using RevMan Ver 5.3 Cochrane Collaboration. Results From 11,747 studies, 117 met the inclusion criteria (n = 100,257; MSI-H n = 12,263, (MSI-H 12.2%). Overall, MSI was associated with improved OS (OR 0.80 (0.71, 0.91). When stratified by stage, there was no difference in OS in stage I and IV, but a protective effect in stage II (OR 0.69 (0.51, 0.92)) and III (0.70 (0.54, 0.91)) CRC. By age, there was benefit in studies where reported median age Conclusions MSI is associated with improved overall survival in stage II and III colorectal cancer. Improved prognosis is seen in younger patients, in both right and left sided colon cancer, but evidence is limited in rectal cancer. MSI was associated with less relapse, but was not associated with cancer specific survival at any stage. Legal entity responsible for the study The authors. Funding Has not received any funding. Disclosure All authors have declared no conflicts of interest.

Circulating tumor cells (CTCs) are now routinely isolated from blood, and measurement of CTC concentrations appears to correlate well with survival in patients with cancer. Interrogation of the molecular profile of CTCs for expression of protein biomarkers, genetic variants and gene expression provides opportunities to use this information to guide personalized treatment, monitor therapy and detect emerging resistance. However, successful application of profiling techniques requires analyses that deliver a reliable and clinically relevant representation of a patient's cancer as it changes with time. Here, we comprehensively review the current knowledge of therapeutically relevant biomarkers in isolated CTCs obtained by fluorescence imaging and genomic profiling approaches. The reviewed data support the notion that molecular profiling of CTCs will provide a reliable representation or surrogate index of tumor burden. Large-scale translational trials, many currently in progress, will provide critical data to progress CTC analysis toward wider clinical use in personalized treatment.

Background Despite an extensive literature suggesting that high microsatellite instability (MSI-H) enhances survival and protects against recurrence after colorectal cancer resection, such effects remain controversial as many studies show only a weak bivariate association or no multivariable association with outcome. This study examined the relationship between MSI status and colorectal cancer outcomes with adjustment for death from other causes as a competing risk. Methods A hospital database of patients following colorectal cancer resection was interrogated for clinical, operative, pathology, adjuvant therapy and follow-up information. MSI-H status was determined by immunohistochemistry for mismatch repair protein deficiency. The cumulative incidence of recurrence and colorectal cancer-specific death was evaluated by competing risks methods. Results Among 1009 patients who had a resection between August 2002 and December 2008, and were followed to at least December 2013, there were 114 (11·3 per cent) with MSI-H (72·8 per cent aged at least 70 years; 63·2 per cent women). After potentially curative resection, with adjustment for non-colorectal cancer death as a competing risk and adjustment for 22 clinical, operative and pathological variables, there was no association between MSI-H and recurrence (hazard ratio (HR) 0·81, 95 per cent c.i. 0·42 to 1·57) or colorectal cancer-specific death (HR 0·73, 0·39 to 1·35) in this patient population. For palliative resections, there was no association between MSI-H and colorectal cancer-specific death (HR 0·65, 0·21 to 2·04). MSI-H was associated with non-colorectal cancer death after both curative (HR 1·55, 1·04 to 2·30) and palliative (HR 3·80, 1·32 to 11·00) resections. Conclusion Microsatellite instability status was not an independent prognostic variable in these patients.

// Tia Morosin 1 , Bruce Ashford 2, 3, 4, 5 , Marie Ranson 2, 3, 5 , Ruta Gupta 6, 7 , Jonathan Clark 8, 9, 10, 11 , N. Gopalakrishna Iyer 12 , Kevin Spring 5, 10, 13 1 Graduate School of Medicine, University of Wollongong, Wollongong, NSW 2522, Australia 2 School of Biological Sciences, University of Wollongong, Wollongong, NSW 2522, Australia 3 Illawarra Health and Medical Research Institute (IHMRI), Wollongong, NSW 2522, Australia 4 Illawarra and Shoalhaven Local Health District (ISLHD), Wollongong, NSW 2522, Australia 5 Centre for Oncology Education and Research Translation (CONCERT), Liverpool, NSW 2170, Australia 6 Department of Tissue Pathology and Diagnostic Oncology, Royal Prince Alfred Hospital, Camperdown, NSW 2050, Australia 7 The University of Sydney, Sydney, NSW 2006, Australia 8 Sydney Head and Neck Cancer Institute, Department of Head and Neck Surgery, The Chris O'Brien Lifehouse, Camperdown, NSW 2050, Australia 9 Central Clinical School, The University of Sydney, Sydney, NSW 2006, Australia 10 South West Clinical School, Faculty of Medicine, The University of New South Wales, Sydney, NSW 2052, Australia 11 Singhealth/Duke-NUS Head and Neck Center, National Cancer Center Singapore (NCCS), 169610, Singapore 12 Medical Oncology Group, Ingham Institute for Applied Medical Research, Liverpool, NSW 2170, Australia 13 Liverpool Clinical School, Western Sydney University, Liverpool, NSW 1871, Australia Correspondence to: Bruce Ashford, email: bgashford@gmail.com Keywords: circulating tumour cells (CTCs), metastases, cutaneous, squamous cell carcinoma (SCC), head and neck Received: February 25, 2016 Accepted: April 16, 2016 Published: June 11, 2016 ABSTRACT Background: Circulating tumour cells (CTCs) are increasingly being used in the surveillance of cancer, allowing for potential early detection and real-time monitoring of disease progression. The presence of CTCs in patients with metastatic cutaneous head and neck squamous cell carcinoma (cHNSCC) has not been evaluated. Results: CTCs were detected in eight of ten patients with regional metastatic cHNSCC (80%; range 1–44 cells/9 mL blood). CTMs were detected in three of ten patients (30%, range 1–4 cells/9 mL blood). Methods: Preoperative blood samples from ten patients with nodal metastases from cutaneous squamous cell carcinomas (cSCC) were analyzed using the IsoFlux TM System for the detection and enumeration of CTCs and circulating tumour microemboli (CTMs). Conclusions: For the first time CTCs have been detected in patients with nodal metastases from cHNSCC. Further work is required to understand their prognostic significance and potential to directly influence clinical practice.

Sporadic colorectal cancers with a high degree of microsatellite instability are a clinically distinct subgroup with a high incidence of BRAF mutation and are widely considered to develop from serrated polyps. Previous studies of serrated polyps have been highly selected and largely retrospective. This prospective study examined the prevalence of sessile serrated adenomas and determined the incidence of BRAF and K-ras mutations in different types of polyps.An unselected consecutive series of 190 patients underwent magnifying chromoendoscopy. Polyp location, size, and histologic classification were recorded. All polyps were screened for BRAF V600E and K-ras codon 12 and 13 mutations.Polyps were detected in 72% of patients. Most (60%) were adenomas (tubular adenomas, tubulovillous adenomas), followed by hyperplastic polyps (29%), sessile serrated adenomas (SSAs; 9%), traditional serrated adenomas (0.7%), and mixed polyps (1.7%). Adenomas were more prevalent in the proximal colon (73%), as were SSAs (75%), which tended to be large (64%5 mm). The presence of at least one SSA was associated with increased polyp burden (5.0 vs 2.5; P.0001) and female sex (P.05). BRAF mutation was rare in adenomas (1/248 [0.4%]) but common in SSAs (78%), traditional serrated adenomas (66%), mixed polyps (57%), and microvesicular hyperplastic polyps (70%). K-ras mutations were significantly associated with goblet cell hyperplastic polyps and tubulovillous adenomas (P.001).The prevalence of SSAs is approximately 9% in patients undergoing colonoscopy. They are associated with BRAF mutation, proximal location, female sex, and presence of multiple polyps. These findings emphasize the importance of identifying and removing these lesions for endoscopic prevention of colorectal cancer.

Background & Aims: Recently, an alternative pathway of tumorigenesis has been identified in the colorectum associated with serrated precursor lesions, variable levels of microsatellite instability (MSI-V), and driven in part by activating mutations in the BRAF proto-oncogene (V599E). Somatic BRAF mutations in hereditary nonpolyposis colon cancer (HNPCC) are rarely observed. Here, we discuss their role in the development of other familial colorectal cancers (CRC). We studied non-FAP, non-HNPCC CRC families characterized by tumors that varied in their level of MSI between individual members. Methods: A subset of tumors from a total of 55 collected (25 polyp and 30 cancers) from 43 individuals across 11 families underwent pathology review, examination for V599E using allele-specific polymerase chain reaction, and for methylation of the MINT31 CpG island. Results: All MSI-V families met the current revised Bethesda Guidelines and 6 of 11 (55%) met the Amsterdam I criteria. V599E was observed in 12 of 19 (63%) polyps and 14 of 20 (70%) cancers (4 of 4 high MSI, 2 of 4 low MSI, and 8 of 12 stable MSI), a significant increase over HNPCC (0 of 15 or 0%), and unselected CRC (30 of 197 or 15.2%) (P < .05). Eight of the 10 (80%) cancers that underwent analysis showed hypermethylation of MINT31. CRCs showed early age at onset and were more likely to show a serrated architecture than unselected CRCs (P < .05). Conclusion: These data provide evidence that the families described here represent a syndrome of familial CRC that is distinct from HNPCC. High levels of BRAF mutation and MINT31 hypermethylation suggest an origin in the serrated pathway of CRC development.

e15081 Background: Changes in microRNA (miRNA) expression during treatment for locally advanced rectal cancer (LARC) may provide insight into disease biology and potentially act as predictive biomarkers. We investigated 112 miRNAs in serum during neoadjuvant chemoradiation for LARC. Methods: Serum was collected at baseline, week 3 and at completion of chemoradiation from 40 prospectively recruited patients with LARC. Responders were classed as tumour regression grade (AJCC classification) 0 or 1 and non-responders as 2 or 3. Serum was also collected from 20 healthy controls. RNA extraction was performed using the Norgen total RNA purification kit. Reverse transcription and pre-amplification were performed according to Taqman OpenArray MicroRNA Panels manufacturer's instructions. QuantStudio12K platform was used for miRNA array qPCR. The delta-delta-Ct method was used to identifiy differentially expressed miRNAs, normalised against U6 snRNA. Analysis was performed in R using paired t-statistics and the Benjamini-Hochberg False Discovery Rate for multiple hypothesis testing adjustment, with q < 0.05 for significance. Enriched KEGG pathways were identified using DIANA, based on verified gene targets of each miRNA from Tarbase. Results: Four miRNAs (miR-125b-1, miR-1183, miR-130a, miR-375) were differentially expressed in baseline patient samples compared to controls. From baseline to completion of treatment, three of these - miR-125b-1, miR-1183, miR-130a were downregulated by more than 2-fold. Comparing responders and non-responders, miR-130a was significantly downregulated in the non-responders only. Conclusions: miR-125b-1, miR-1183 and miR-130a are significantly downregulated in patients with LARC during chemoradiation. These miRNAs are known to target key colorectal cancer genes such as ATM and CHEK1, which have been implicated in chemoradiation resistance. miR-130a warrants further investigation as a predictive biomarker, being downregulated in patients with a poor response to therapy. Work is ongoing, investigating these miRNA targets in the solid tissue in these patients. To our knowledge, this is the first study to profile potentially predictive miRNA changes during chemoradiation in LARC.

Locally advanced rectal cancer is regularly treated with trimodality therapy consisting of neoadjuvant chemoradiation, surgery and adjuvant chemotherapy. There is a need for biomarkers to assess treatment response, and aid in stratification of patient risk to adapt and personalise components of the therapy. Currently, pathological stage and tumour regression grade are used to assess response. Experimental markers include proteins involved in cell proliferation, apoptosis, angiogenesis, the epithelial to mesenchymal transition and microsatellite instability. As yet, no single marker is sufficiently robust to have clinical utility. Microarrays that screen a tumour for multiple promising candidate markers, gene expression and microRNA profiling will likely have higher yield and it is expected that a combination or panel of markers would prove most useful. Moving forward, utilising serial samples of circulating tumour cells or circulating nucleic acids can potentially allow us to demonstrate tumour heterogeneity, document mutational changes and subsequently measure treatment response.

Circulating tumour cells (CTCs) are emerging as important prognostic markers and have potential clinical utility as tumour biomarkers for targeted cancer therapy. Although CTCs were proposed more than 100 years ago as potential precursors that may form metastatic lesions, formal evidence that CTCs are indeed capable of initiating metastases is limited. Moreover, the process of CTCs shedding into the circulation, relocating to distant organ sites and initiating metastatic foci is complex and intrinsically inefficient. To partially explain the metastatic process, the concepts of CTCs as metastatic precursors or pre-metastatic conditioners have been proposed; however, it is questionable as to whether these are both variable pathways to metastasis or just markers of metastatic burden. This review explores the evidence for CTCs in the initiation and progression of metastatic cancer and the data supporting these different concepts in an attempt to better understand the role of CTCs in metastasis. A greater understanding of the metastatic potential of CTCs will open new avenues for therapeutic interventions in the future.

Colorectal cancers arising via the serrated pathway are often associated withBRAFV600E mutation, CpG island methylator phenotype (CIMP), and microsatellite instability. Previous studies have shown a strong association betweenBRAFV600E mutation and serrated polyps. This study aims to evaluate CIMP status of all the serrated polyp subtypes and its association with functionally important genes such asMLH1, p16,andIGFBP7. CIMP status and methylation were evaluated using the real-time based MethyLight assay in 154 serrated polyps and 63 conventional adenomas. Results showed that CIMP-high serrated polyps were strongly associated withBRAFmutation and proximal colon. CIMP-high was uncommon in conventional adenomas (1.59%), occurred in 8.25% of hyperplastic polyps (HPs), and became common in sessile serrated adenomas (SSAs) (51.43%).MLH1methylation was mainly observed in the proximal colon and was significantly associated withBRAFmutation and CIMP-high. The number of samples methylated forp16andIGFBP7was the highest in SSAs. The methylation panel we used to detect CIMP is highly specific for CIMP-high cancers. With this panel, we demonstrate that CIMP-high is much more common in SSAs than HPs. This suggests that CIMP-high correlates with increased risk of malignant transformation which was also observed in methylation of functionally important genes.

There is increasing evidence for the use of circulating tumor cells (CTCs) as a "liquid biopsy" for early detection of lung cancer recurrence, prognosticating disease and monitoring treatment response. Further, CTC molecular analysis and interrogation of single cells hold significant potential in providing insights into tumor biology and the metastatic process. Ongoing research will likely see the translation of CTCs as a prognostic and predictive biomarker in both small cell, and non-small cell, lung cancer to routine clinical practice.

Introduction MicroRNAs (miRNA) expression may change with treatment and influence responses in locally advanced rectal cancer (LARC) making them potentially useful predictive and prognostic biomarkers. We investigated 112 miRNAs in serum during chemoradiation for LARC. Methods Twenty-one patients with LARC treated with neoadjuvant chemoradiation were prospectively recruited for the study. Serum was collected at three time-points: 1) at diagnosis (baseline), 2) at three weeks into treatment and 3) at completion of chemoradiation (pre-surgery). Serum was also collected from 10 healthy controls. RNA extraction was performed using the Norgen™ total RNA purification kit. Reverse transcription and pre-amplification were performed as per the Taqman™ OpenArray MicroRNA Panels manufacturer's instructions. miRNA array qPCR was performed on 112 miRNA targets using the QuantStudio™ 12K platform. Differentially expressed miRNAs were identified using the delta-delta-Ct method, using the endogenous U6 snRNA as the control. Analysis was performed in R, using paired t-statistics, and the Benjamini-Hochberg False Discovery Rate for multiple hypothesis testing adjustment, with a threshold q Results We identified 12 miRNAs that were significantly differentially expressed between patients and matched controls. The strongest differences were observed between patient samples at baseline and completion of chemoradiation, where eight miRNAs decreased in expression: hsa-miR-101, hsa-miR-130a, hsa-miR-130b, hsa-miR-223, hsa-miR-27a, hsa-miR-628-5p, hsa-miR-630 and hsa-miR-720, all with fold change > 3-fold. These genes are known to target key rectal cancer genes such as SMAD4, BCL2, MSH2 and TGFBR2. An additional three miRNAs changed significantly between baseline and week 3: hsa-miR-135b*, hsa-miR-375 and hsa-miR-629. All except hsa-miR-135b* became less abundant. Conclusions Ten differentially expressed miRNAs appear downregulated during chemoradiation in LARC. These miRNAs have been implicated in cell proliferation, the epithelial-mesenchymal transition and radiation resistance. Further work will be undertaken to understand the functional implications of these changes. Citation Format: Stephanie H. Lim, Paul De Souza, Wei Chua, Weng Ng, Benjamin Harris, Mark J. Cowley, Kevin Spring. Change in serum microRNA expression during neoadjuvant chemoradiation for rectal cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1942.

This review explores the chief genetic and epigenetic events that promote pathological progression in colorectal carcinogenesis. This article discusses the molecular and pathological basis for classifying colorectal neoplasia into suppressor, mutator and methylator pathways. These differing mechanisms of genomic instability are associated with specific cancer characteristics, and may provide the opportunity for more effective prevention and surveillance strategies in the future. This is the first review in a series of five topics outlining important and developing aspects of colorectal cancer.

e14542 Background: Human polo-like kinase 1 (PLK1) is important in mitotic progression. It is overexpressed in many cancers including colorectal cancer. Decreased PLK1 expression has been associate...

Sessile serrated polyps (SSPs) are one of the precursors for colon cancer, because of a high BRAF mutation rate. However, the colonoscopic appearances of SSP are still unclear. This study aimed to clarify colonoscopic features of SSPs. Methods: One experienced colonoscopist performed chromocolonoscopy for 190 patients in Australia with the aim to remove all polyps. 166 serrated polyps were detected, of which 54 serrated polyps measuring 5 mm or greater were enrolled. Polyp size was assessed based on resected specimens. One independent colonoscopist reviewed printed photographs of the serrated polyps, blinded to histology. Polyp morphology (flat, protruded), polyp surface (smooth, rough), polyp hue (pale, pink), adherent mucus (thin, thick) and pit pattern (asteroid, elliptic, gyrus-like) were evaluated. Histology was assessed by a single pathologist, and serrated polyps were subclassified as goblet cell (GC) type, microvesicular (MV) type, SSP, traditional serrated adenoma (TSA) and mixed polyp. Results: Ten serrated polyps were excluded because of poor quality photographs, leaving 44 serrated polyps which were studied. The mean size ( ± SD) of the 44 serrated polyps was 8.8 ± 4.0 mm, and 61% were located in the proximal colon. The mean size, location and colonoscopic features of each histological type are shown in the table. All 5 mixed polyps included a TSA component on histology, so these types were combined. The mean size of polyps in the TSA/mixed polyp category was significantly larger than those of MV type and SSP (Bonferroni correction), but there was no significant difference between mean size of MV type and SSP. Except for proximal location, SSPs were very similar in endoscopic appearance to the MV type. Both types of serrated polyps were pale, smooth and flat in appearance with an asteroid pit pattern. The majority of GC type located in the proximal colon showed protruded morphology. The TSA and mixed polyps were distributed evenly and there was no specific morphology. Conclusion: At colonoscopy, GC type can be discriminated from MV type and SSP. However, both MV type and SSP were pale, smooth and flat in appearance with an asteroid pit pattern, making differentiation difficult. The only difference was the anatomical distribution.