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Background First‐line platinum‐based therapy for advanced squamous cell carcinomas of the anal canal (SCCA) implies a risk of substantial side effects, and data on second‐line treatment options are limited. Paclitaxel and Capecitabine are a well‐known regimen with a moderate toxicity profile, but its efficacy has not been evaluated. Methods We conducted a retrospective study using Danish Hospital Registers of patients treated with Paclitaxel and Capecitabine for inoperable, recurrent, or advanced metastatic SCCA in Denmark, between January 2000 and July 2018. Results A total of 52 patients met the eligibility criteria. Median age was 60.7 years (range 42–83). Efficacy was observed, with an overall response rate in patients receiving first‐line (N = 28) and second‐line (N = 23) Paclitaxel and Capecitabine of 39.3% (2 with complete responses) and 17.4%, respectively. Median progression‐free survival (PFS) was 4.5 months (95% CI 3.3–5.9) and 3.8 months (95% CI 2.4–5.5) with OS of 6.7 months (95% CI 5.9–8.5) and 5.9 months (95% CI 3.9–14), respectively. Performance status ≥2 and neutrophil to lymphocyte ratio ≥4 were significantly associated with a short PFS. Conclusion This study recognizes Paclitaxel and Capecitabine as a potential regimen for advanced SCCA, when recommended first‐line therapy is not feasible or as a potential second‐line treatment after failure of platinum‐based chemotherapy., Paclitaxel and Capecitabine is recognized for advanced anal cancer when recommended first‐line therapy is not feasible or as second‐line treatment after failure of platinum‐based chemotherapy.

Simple Summary The primary treatment modality for anal cancer is chemoradiotherapy, but patients with locally advanced disease (i.e., large tumors and/or involvement of regional lymph nodes) have a high risk of treatment failure. The use of chemotherapy prior to radiotherapy (induction chemotherapy) can potentially shrink the tumor and/or eradicate small cancer cells with metastatic potential, with a chance of a better outcome. With this paper, the authors present 20 years of nationwide experience with intensified induction chemotherapy in the treatment of locally advanced anal cancer, which indicates a role for further investigation in the most advanced cases. Abstract Locally advanced squamous cell carcinoma of the anus (LASCCA) has a poor prognosis with a high risk of treatment failure calling for intensified therapy. We present the long-term follow-up of a nationwide cohort of LASCCA treated with intensified induction chemotherapy (ICT). The study included patients with LASCCA (T3-4N0 or T1-4N+) treated with at least one cycle of ICT (cisplatin, ifosfamide, leucoverin, and 5-flourouracil) between 1998–2018. Data were retrospectively collected from medical records, and statistics were performed in STATA 16.1. In total, 166 patients with LASCCA were identified. Following ICT, 157 patients (95%) received primary curative treatment with either radiotherapy (70%), chemoradiotherapy (27%), or abdominal perineal resection (3%). The overall local tumor response rate after ICT was 76% with 20 (13%) achieving complete local tumor response. After the primary treatment, 123 patients (79%) obtained complete response, and 27 underwent salvage surgery due to persistent disease. The median follow-up time was 6 years, local and distant failure rates 22% and 13%, respectively. The 3- and 5-year disease-free survival rates were 70% and 67%, and the 3- and 5-year overall survival rates were 76% and 70%, respectively. Intensified ICT regimen could be a supplementary treatment option in the most advanced cases of LASCCA. Prospective randomized trials are needed to investigate this approach further.

Re‐exposure to immunotherapy in metastatic colorectal cancer may be indicated in selected patients that previously benefitted from immunotherapy with tolerable irAEs.

e15567 Background: Precision oncology based on in-vitro, functional assays has potential advantages compared to the much more common molecular approach, but the clinical benefit is unknown. We here report the results from the largest prospective interventional clinical trial testing the clinical outcome in colorectal cancer patients treated with drugs showing cytotoxic effect in matched patient-derived tumoroids. Methods: This single-center, phase II trial included patients with metastatic colorectal cancer previously exposed to all standard therapies. Specimens from one to three 18-16 G core needle biopsies were manually dissected, enzymatically treated, cultivated, and incubated to form 3D spherical microtumors, i.e. tumoroids. In the assay for in-vitro sensitivity testing, the tumoroids were challenged with single drugs and combinations thereof to determine patient-specific responses. Using tumoroid screening technology (IndiTreat, 2cureX, Copenhagen, Denmark), results were generated by comparing the sensitivity of the individual patient’s tumoroids with a reference panel from other patients. The testing included standard cytostatics and drugs with proven effect in previous early-phase clinical trials, a total of 15 drugs. The primary endpoint was the fraction of patients with progression-free survival (PFS) at two months. Based on placebo arms in randomized last-line trials, a minimal relevant difference of 20% (20% to 40%) was stated. Using Simon's two-stage design, a sample size of 45 patients was calculated with at least 14 PFS at two months (significance 5%, power 90%). Results: Ninety patients were enrolled from 9/2017 to 9/2020. Biopsies from 82 patients were obtained and sent for tumoroid formation of which 44 (54%, 95% CI 42-65) were successful and at least one treatment was suggested. Thirty-four patients initiated treatment according to the response obtained in the drug assays within a median of 51 days from inclusion (IQR 39-63). The primary endpoint, PFS at two months, was met in 17 of 34 patients (50%, 95%CI 32-68). There were no radiological responses. Median PFS was 81 days (95% CI 51-112) and median OS was 189 days (95% CI 103-277). Conclusions: Precision oncology using a functional approach with patient-derived tumoroids and in-vitro drug sensitivity testing seems feasible. The approach is limited by the fraction of patients with successful tumoroid development. The primary endpoint was met, as half of the patients were without progression at two months. Further clinical studies are justified. Clinical trial information: NCT03251612.

The treatment for locally advanced rectal carcinomas (LARC) is based on neoadjuvant chemoradiotherapy (nCRT) and surgery, which results in pathological complete response (pCR) in up to 30% of patients. Since epigenetic changes may influence response to therapy, we aimed to identify DNA methylation markers predictive of pCR in LARC patients treated with nCRT. We used high-throughput DNA methylation analysis of 32 treatment-na&iuml, ve LARC biopsies and five normal rectal tissues to explore the predictive value of differentially methylated (DM) CpGs. External validation was carried out with The Cancer Genome Atlas-Rectal Adenocarcinoma (TCGA-READ 99 cases). A classifier based on three-CpGs DM (linked to OBSL1, GPR1, and INSIG1 genes) was able to discriminate pCR from incomplete responders with high sensitivity and specificity. The methylation levels of the selected CpGs confirmed the predictive value of our classifier in 77 LARCs evaluated by bisulfite pyrosequencing. Evaluation of external datasets (TCGA-READ, GSE81006, GSE75546, and GSE39958) reproduced our results. As the three CpGs were mapped near to regulatory elements, we performed an integrative analysis in regions associated with predicted cis-regulatory elements. A positive and inverse correlation between DNA methylation and gene expression was found in two CpGs. We propose a novel predictive tool based on three CpGs potentially useful for pretreatment screening of LARC patients and guide the selection of treatment modality.

There is a need for biomarkers to improve the clinical benefit from systemic treatment of colorectal cancer. We designed a prospective, clinical study where patients receiving regorafenib as last-line treatment had sequential blood samples drawn. Effect and toxicity was monitored. The primary clinical endpoint was progression free survival (PFS). Cell-free circulating tumor (ct) DNA was measured as either the fraction with Neuropeptide Y (NPY) methylated DNA or KRAS/NRAS/BRAF mutated ctDNA. One hundred patients were included from three Danish centers. Among 95 patients who received regorafenib for at least two weeks, the median PFS was 2.1 months (95% confidence interval (CI) 1.8&ndash, 3.3) and the median overall survival (OS) was 5.2 months (95% CI 4.3&ndash, 6.5). Grade 3&ndash, 4 toxicities were reported 51 times, most frequently hypertension, hand-food syndrome, and skin rash. In the biomarker population of 91 patients, 49 could be monitored using mutated DNA and 90 using methylated DNA. There was a strong correlation between mutated and methylated DNA. The median survival for patients with a level of methylated ctDNA above the median was 4.3 months compared to 7.6 months with ctDNA below the median, p &lt, 0.001. The median time from increasing methylated ctDNA to disease progression was 1.64 months (range 0.46&ndash, 8.38 months). In conclusion, NPY methylated ctDNA was a universal liquid biopsy marker in colorectal cancer patients treated with regorafenib. High baseline levels correlated with short survival and changes during treatment may predict early effect and later progression. We suggest plasma NPY methylation analysis as an easy and universally applicable method for longitudinal monitoring of ctDNA in metastatic colorectal cancer patients.

Background Tumour tissue from individual patients with colorectal cancer may be cultured in vitro as three-dimensional tumoroids that resembles the biological features in vivo including chemotherapy sensitivity and resistance. Tumoroid formation is well-established for resected tumour tissue, but there is a lack of detailed descriptions of cultivating tumoroids from liver biopsies. The purpose of this study was to compare metastasis, imaging, biopsy histology and tumoroid yield. Methods Patients exposed to all standard medical treatment for metastatic colorectal cancer were included within an ongoing phase II clinical trial (NCT03251612). Biopsies from liver were obtained with 16G needles and other localizations 18G. Microscopy was used to evaluate necrosis (present or not) and to quantify the fraction of vital carcinoma cells: 0 (0%), 1 (0-10%), 2 (10-50%) or 3 (50-100%). Tumoroid formation was quantified as 0 (0 tumoroids), 1 (1-10), 2 (10-100), 3 (100-1000) or 4 (>1000). Biopsies of low quality were pooled up to three together for tumoroid formation. Elastography during ultrasound were used to measure the tissue stiffness of a subset of liver metastases and was together with metastasis size other explorative variables. Results A total of 78 biopsies from 27 biopsy sessions were included from liver (22), lung (2), carcinomatosis (1), muscle (1) or kidney (1). No serious adverse events related to biopsy procedures were observed. 37 (49%) of 76 biopsies for histology contained vital carcinoma cells and necrosis was present in 43 (57%). The number of tumoroids after cultivation correlated with the level of vital carcinoma cells in biopsies (p = 0.008) and trend-wise inversely with the stiffness of the tumour (p = 0.05) but not the size of metastasis (p = 0.97) nor presence of necrosis (0.98). Conclusions The tumoroid yield after cultivation of biopsies from chemo-refractory colorectal cancer patients correlates with the level of carcinoma cells determined by microscopy and probably with the stiffness of the metastasis. Necrosis and size of metastasis were not related. These findings may have great implications for selecting and evaluating biopsies for tumoroid formation in precision medicine to colorectal cancer patients. Clinical trial identification NCT03251612. Legal entity responsible for the study The authors. Funding 2cureX. Disclosure L.H. Jensen: Research grant / Funding (institution): MSD; Research grant / Funding (institution): BMS; Research grant / Funding (institution): Incyte; Research grant / Funding (institution): 2cureX. G. Hagel: Shareholder / Stockholder / Stock options, Full / Part-time employment: 2cureX. H. Harling: Advisory / Consultancy: 2cureX. O. Thastrup: Leadership role, Shareholder / Stockholder / Stock options, Full / Part-time employment, Officer / Board of Directors: 2cureX. All other authors have declared no conflicts of interest.

Made available in DSpace on 2019-10-06T17:12:46Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-12-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Most patients with locally advanced rectal cancer (LARC) present incomplete pathological response (pIR) to neoadjuvant chemoradiotherapy (nCRT). Despite the efforts to predict treatment response using tumor-molecular features, as differentially expressed genes, no molecule has proved to be a strong biomarker. The tumor secretome analysis is a promising strategy for biomarkers identification, which can be assessed using transcriptomic data. We performed transcriptomic-based secretome analysis to select potentially secreted proteins using an in silico approach. The tumor expression profile of 28 LARC biopsies collected before nCRT was compared with normal rectal tissues (NT). The expression profile showed no significant differences between complete (pCR) and incomplete responders to nCRT. Genes with increased expression (pCR = 106 and pIR = 357) were used for secretome analysis based on public databases (Vesiclepedia, Human Cancer Secretome, and Plasma Proteome). Seventeen potentially secreted candidates (pCR = 1, pIR = 13 and 3 in both groups) were further investigated in two independent datasets (TCGA and GSE68204) confirming their over-expression in LARC and association with nCRT response (GSE68204). The expression of circulating amphiregulin and cMET proteins was confirmed in serum from 14 LARC patients. Future studies in liquid biopsies could confirm the utility of these proteins for personalized treatment in LARC patients. International Research Center - CIPE A.C.Camargo Cancer Center Department of Clinical Genetics University Hospital of Southern Denmark Department of Morphology – Institute of Bioscience São Paulo State University (UNESP) Department of Pelvic Surgery A.C.Camargo Cancer Center Department of Pathology A.C.Camargo Cancer Center Molecular Oncology Research Center Barretos and Diagnósticos da América (DASA) Department of Biochemistry and Immunology University Hospital of Southern Denmark Danish Colorectal Cancer Center South Institute of Regional Health Research Faculty of Health Sciences University of Southern Denmark Department of Oncology University Hospital of Southern Denmark Department of Morphology – Institute of Bioscience São Paulo State University (UNESP) FAPESP: 2008/57887-9 FAPESP: 2014/06323-9 FAPESP: 2015/25803-4 CNPq: 573589/08-9

The aim of the present study was to investigate the influence of fixation delay and the perioperative ischemia on hypoxia inducible factor (HIF)-1α gene expression, HIF-1α protein expression, and immunohistochemical (IHC) expression of HIF-1α, GLUT-1, Bcl-2, and Ki-67 in colorectal cancer. The study included 25 surgically removed colorectal tumors. Three sets of samples were collected readily after removal and exposed to 0, 30, and 60 minutes of delay of fixation or freezing. The perioperative ischemia time was registered. In each set of the samples, HIF-1α gene expression was analyzed by quantitative real time polymerase chain reaction, protein concentration of HIF-1α was assessed by enzyme-linked immunosorbent assay, and IHC staining of HIF-1α, GLUT-1, Bcl-2, and Ki-67 was performed. Preoperative formalin-fixed paraffin-embedded biopsies and whole sections of the entire tumor were analyzed by IHC. We found that the HIF-1α gene expression, HIF-1α protein concentration, and IHC expression of HIF-1α, GLUT-1, Ki-67, and Bcl-2 were not systematically affected by either the fixation or freezing delay of the tissue, the perioperative ischemia time, or the total ischemia time (perioperative ischemia+delay of fixation or freezing) in colorectal tumors. However, the intraindividual variation was quite large, which may question the use of individually, non-standardized-handled single biopsies or small tissue samples for analysis of often rather heterogenously expressed biomarkers.

Objective: The aim of this study was to investigate the feasibility of 18F-fluoroazomycinarabinofuranoside ( 18F-FAZA) positron emission tomography (PET)/computed tomography (CT) in patients with locally advanced rectal cancer. Materials and Methods: The study included 14 patients with locally advanced rectal cancer. Before chemoradiotherapy, PET/CT with F-FAZA was performed with static 15 min images 2 h after injection of 18F-FAZA. Attenuation correction was obtained with a low-dose CT, and a contrast-enhanced CT was performed immediately after the PET scan. Results: 18F-FAZA uptake [mean and maximum standardized uptake value (SUVmean) and (SUVmax)] was significantly higher in rectal tumours than in both muscles (Pmax, SUV mean, T/M and T/I values were significantly higher in the entire tumour volume compared with the tumour volume adjusted for scatter from the urinary bladder (P18F-FAZA PET/CT is feasible for visualization of hypoxia in patients with rectal cancer, but scattered activity from the urinary bladder should be taken into consideration.

e15541Background: Regorafenib has a proven but small significant effect in refractory colorectal cancer. The objective of the present, post-marketing trial was to investigate the clinical outcome i...

Purpose/Objective(s) Mature data on tumor control and survival are presented from a randomized trial of the addition of a brachytherapy boost to long-course neoadjuvant chemoradiation therapy (CRT) for locally advanced rectal cancer. Methods and Materials Between March 2005 and November 2008, 248 patients with T3-4N0-2M0 rectal cancer were prospectively randomized to either long-course preoperative CRT (50.4 Gy in 28 fractions, per oral tegafur-uracil and L-leucovorin) alone or the same CRT schedule plus a brachytherapy boost (10 Gy in 2 fractions). The primary trial endpoint was pathologic complete response (pCR) at the time of surgery; secondary endpoints included overall survival (OS), progression-free survival (PFS), and freedom from locoregional failure. Results Results for the primary endpoint have previously been reported. This analysis presents survival data for the 224 patients in the Danish part of the trial. In all, 221 patients (111 control arm, 110 brachytherapy boost arm) had data available for analysis, with a median follow-up time of 5.4 years. Despite a significant increase in tumor response at the time of surgery, no differences in 5-year OS (70.6% vs 63.6%, hazard ratio [HR] = 1.24, P =.34) and PFS (63.9% vs 52.0%, HR=1.22, P =.32) were observed. Freedom from locoregional failure at 5 years were 93.9% and 85.7% (HR=2.60, P =.06) in the standard and in the brachytherapy arms, respectively. There was no difference in the prevalence of stoma. Explorative analysis based on stratification for tumor regression grade and resection margin status indicated the presence of response migration. Conclusions Despite increased pathologic tumor regression at the time of surgery, we observed no benefit on late outcome. Improved tumor regression does not necessarily lead to a relevant clinical benefit when the neoadjuvant treatment is followed by high-quality surgery.

Background. Patients with chemotherapy refractory metastatic colorectal cancer and KRAS mutations have no effective treatment option. The present study evaluated the efficacy of temsirolimus in chemotherapy refractory mCRC with KRAS mutations. Furthermore, we wanted to investigate if resistance to temsirolimus could be reversed by the addition of irinotecan. Finally, we analyzed pre-treatment blood samples for KRAS mutations to investigate the association between quantitative measures of KRAS mutated alleles and clinical outcome. Material and methods. Patients received weekly temsirolimus 25 mg until progression. Thereafter patients were treated with combination therapy comprising biweekly irinotecan 180 mg/m(2) and weekly temsirolimus. A polymerase chain reaction method was used to quantify the KRAS mutated alleles in plasma (pKRAS). Results. Sixty-four patients were included. Treatment was well tolerated. Thirty-eight percent achieved stable disease on monotherapy and 63% on combination therapy. Four and eight patients had a minimal response, respectively. Median overall survival was 160 days. Median time to progression was 45 and 84 days, respectively. The concordance between KRAS status in tumor and plasma was 82%. All patients with tumor reduction had low levels of pKRAS. Patients with high pKRAS had a 77% risk of early progression on monotherapy compared to 43% in patients with lower levels. Multivariate survival analysis confirmed that pKRAS was a strong prognostic factor. Conclusion. Temsirolimus has limited efficacy in chemotherapy resistant KRAS mutant disease, but plasma KRAS quantification is a strong predictor of outcome.

The aim of this study was to investigate the predictive impact of polymorphisms in the HIF-1α gene on the response to chemoradiotherapy (CRT) in rectal cancer. This study included two cohorts of patients with locally advanced rectal cancer receiving long-course CRT. The HIF-1α C1772T (rs11549465), G1790A (rs11549467) and c(*)191T>C (rs2057482) polymorphisms were investigated in the test cohort (n=65), and HIF-1α c(*)191T>C was analysed in the validation cohort (n=198). No correlations were identified between the polymorphisms and clinicopathological factors. The HIF-1α C1772T and HIF-1α G1790A polymorphisms demonstrated no correlation with tumour response to CRT in the test cohort. The HIF-1α c(*)191T>C CC genotype was marginally associated with a higher rate of complete tumour response (P=0.05) in the test cohort, while the HIF-1α c(*)191T>C CC genotype was associated with a poor tumour response (P=0.03) in the validation cohort. In conclusion, these results suggest that HIF-1α polymorphisms have no value as predictors of response to neoadjuvant CRT in rectal cancer. The results of the HIF-1α c(*)191T>C in two cohorts differ and emphasise the importance of biomarker validation.

Introduction Tumour hypoxia is well-established phenomenon in solid tumours and is an important factor in relation to radioresistance. Visualising hypoxia by positron emission tomography (PET) is attractive because it is a non-invasive method, and leaves an option to measure hypoxia consecutively during treatment. 18F-Flouroazomycinarabinofuranoside (18F-FAZA) has attractive pharmacokinetic properties. In experimental models 18F-FAZA is accumulated in hypoxic tumour cells and rapidly cleared from non-hypoxic tissues. Only few studies are available of 18F-FAZA PET/CT scans of human tumours and none of these include patients with rectal cancer. The aim of this study was to investigate the feasibility of 18F-FAZA PET/CT in patients with locally advanced rectal cancer. Methods The study included 14 patients with locally advanced rectal cancer (cT3-T4, cN1-2, cM0, CRM Results The rectal tumours had a median value (range) of SUVmax 2.06 (1.23-2.99) and SUVmean 1.31 (0.72-2.04). 18F-FAZA uptake (SUVmax and SUVmean) was significantly higher in the rectal tumours compared with both muscle (p Conclusion F-FAZA PET/CT imaging is feasible in patients with rectal cancer visualising hypoxia but scattered activity from the urinary bladder should be taken into consideration.