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Supplementary Table S1. Summary of colorectal cancer patient samples used in this study. The tissue site from which samples originated or were collected, disease stage, and main driver mutations are listed.

Supplementary Materials and Methods (unmarked)

Purpose: Cancer-initiating cells (C-IC) have been described in multiple cancer types, including colorectal cancer. C-ICs are defined by their capacity to self-renew, thereby driving tumor growth. C-ICs were initially thought to be static entities; however, recent studies have determined these cells to be dynamic and influenced by microenvironmental cues such as hypoxia. If hypoxia drives the formation of C-ICs, then therapeutic targeting of hypoxia could represent a novel means to target C-ICs.Experimental Design: Patient-derived colorectal cancer xenografts were treated with evofosfamide, a hypoxia-activated prodrug (HAP), in combination with 5-fluorouracil (5-FU) or chemoradiotherapy (5-FU and radiation; CRT). Treatment groups included both concurrent and sequential dosing regimens. Effects on the colorectal cancer-initiating cell (CC-IC) fraction were assessed by serial passage in vivo limiting dilution assays. FAZA-PET imaging was utilized as a noninvasive method to assess intratumoral hypoxia.Results: Hypoxia was sufficient to drive the formation of CC-ICs and colorectal cancer cells surviving conventional therapy were more hypoxic and C-IC-like. Using a novel approach to combination therapy, we show that sequential treatment with 5-FU or CRT followed by evofosfamide not only inhibits tumor growth of xenografts compared with 5-FU or CRT alone, but also significantly decreases the CC-IC fraction. Furthermore, noninvasive FAZA-PET hypoxia imaging was predictive of a tumor's response to evofosfamide.Conclusions: Our data demonstrate a novel means to target the CC-IC fraction by adding a HAP sequentially after conventional adjuvant therapy, as well as the use of FAZA-PET as a biomarker for hypoxia to identify tumors that will benefit most from this approach. Clin Cancer Res; 24(9); 2116–27. ©2018 AACR.

Supplementary Figure S1. Hypoxia increases stem gene expression and stabilizes HIF proteins. Supplementary Figure S2. Chemotherapy enriches for colorectal C-IC markers and increases expression of Wnt and HIF targets. Supplementary Figure S3. Growth of colorectal cancer PDXs shown in Figure 4. Supplementary Figure S4. Hypoxic fraction in a panel of colorectal cancer PDX models. Supplementary Figure S5. Growth of colorectal cancer PDXs shown in Figure 5. Supplementary Figure S6. Hypoxia gene sets are prognostic for clinical outcome of colon cancer patients.

A 70-year old man had multiple lung nodules detected on a computed tomography (CT) performed as part of the work-up for an isolated episode of hematuria of several hours' duration. A CT-guided lung biopsy disclosed a morphologically undifferentiated neoplasm, that could not be further subclassified despite an extensive immunohistochemical panel.Four years previously the patient had undergone a left hemicolectomy in another institution to resect a 4.7 cm polypoid ulcerated area in the splenic flexure. The slides from that resection were retrieved for review at our institution. There was a variably cellular, diffuse spindle cell neoplastic proliferation involving the submucosa, muscularis propria and subserosa of the large bowel, composed of elongated spindle cells, with small, inconspicuous nucleoli, arranged in intersecting fascicles. Occasional areas of the tumor showed myxoid stroma. Mitotic activity was low.Immunohistochemistry showed strong and diffuse cytoplasmic staining for MUC4 in both the large bowel and lung tumors. Molecular profiling identified a FUS-CREB3L1 fusion gene.The constellation of morphological, immunohistochemical and molecular features are of low-grade fibromyxoid sarcoma (LGFMS) of the large bowel, with metastatic fibromyxoid sarcoma to the lung.LGFMS is a malignant, often late-metastasizing tumor, with a misleadingly bland histological appearance, that typically arises in the deep soft tissues of the proximal extremities or trunk of young adults. Accurate classification, requiring morphology and ancillary tests, is required for appropriate clinical follow-up and treatment of patients. Keywords: Colon, Low-grade fibromyxoid sarcoma, Metastasis, Lung

This study aimed to ascertain views, incidence of reporting and diagnostic criteria for gastric foveolar dysplasia. A questionnaire, a post-questionnaire discussion and microscopic assessment of selected cases was conducted by gastrointestinal pathologists to explore the above-stated aims. Fifty-four percent of respondents never or rarely diagnosed gastric foveolar-type dysplasia. The general consensus was that round nuclei, lack of nuclear stratification, presence of inflammation/damage and surface maturation favoured reactive change; while architectural abnormalities/complexity and nuclear enlargement mainly were used to separate low-grade from high-grade foveolar dysplasia. Immunohistochemistry was rarely used to make the diagnosis of dysplasia and was thought not to be of help in routine practice. Inter-observer agreement in grading of dysplasia versus reactive, and the type of dysplasia (foveolar versus adenomatous), was substantial/almost perfect amongst 35.7% and 21.4% of participants, respectively. This reflects low reproducibility in making these diagnoses. In conclusion, foveolar dysplasia was a rarely made diagnosis among 14 gastrointestinal pathologists, there are no uniform criteria for diagnosis and there is poor inter-observer agreement in separating low-grade foveolar dysplasia from reactive gastric mucosa and low-grade adenomatous dysplasia. Greater awareness and agreed criteria will prevent misdiagnosis of low-grade foveolar dysplasia as reactive, and vice versa.

Granular cell tumour (GCT) is a rare, usually benign, mesenchymal lesion that can occur in nearly any organ, but has a proclivity for the oral cavity, skin and subcutaneous tissue. They occasionally arise in the gastrointestinal tract (GIT) where the oesophagus is most commonly involved. Occurrence in the colorectum is unusual. We report a case of a 73 year-old man with an incidentally discovered GCT with extensive sclerosis in the caecum. Microscopically, a well-circumscribed, paucicellular nodule was identified in the submucosa with prominent hyalinisation and focal myxoid change. Scattered, finely granular cells expressing S100, inhibin and calretinin were identified. CD117 and DOG-1 were negative. This case was made challenging by the prominent sclerosis with only focally visible granular cells, however, the overall features were those of a GCT of the colon. We highlight the morphological and immunohistochemical features of this rare entity in the colon, and discuss the differential diagnosis. In this era where incidental polyps and submucosal lesions are increasingly being detected by endoscopy, knowledge and recognition of GCT in the colon is important as they are generally benign with a good prognosis following complete excision.

Purpose: Cancer-initiating cells (C-IC) have been described in multiple cancer types, including colorectal cancer. C-ICs are defined by their capacity to self-renew, thereby driving tumor growth. C-ICs were initially thought to be static entities; however, recent studies have determined these cells to be dynamic and influenced by microenvironmental cues such as hypoxia. If hypoxia drives the formation of C-ICs, then therapeutic targeting of hypoxia could represent a novel means to target C-ICs. Experimental Design: Patient-derived colorectal cancer xenografts were treated with evofosfamide, a hypoxia-activated prodrug (HAP), in combination with 5-fluorouracil (5-FU) or chemoradiotherapy (5-FU and radiation; CRT). Treatment groups included both concurrent and sequential dosing regimens. Effects on the colorectal cancer-initiating cell (CC-IC) fraction were assessed by serial passage in vivo limiting dilution assays. FAZA-PET imaging was utilized as a noninvasive method to assess intratumoral hypoxia. Results: Hypoxia was sufficient to drive the formation of CC-ICs and colorectal cancer cells surviving conventional therapy were more hypoxic and C-IC-like. Using a novel approach to combination therapy, we show that sequential treatment with 5-FU or CRT followed by evofosfamide not only inhibits tumor growth of xenografts compared with 5-FU or CRT alone, but also significantly decreases the CC-IC fraction. Furthermore, noninvasive FAZA-PET hypoxia imaging was predictive of a tumor's response to evofosfamide. Conclusions: Our data demonstrate a novel means to target the CC-IC fraction by adding a HAP sequentially after conventional adjuvant therapy, as well as the use of FAZA-PET as a biomarker for hypoxia to identify tumors that will benefit most from this approach. Clin Cancer Res; 24(9); 2116–27. ©2018 AACR.

To ascertain the approach and degree of consensus of pathologists in the handling and regression grading of colorectal cancer resection specimens treated with neoadjuvant chemoradiation, a ten-part questionnaire was circulated to 18 gastrointestinal pathologists in eight countries. The questions were specific and addressed pertinent issues related to colorectal cancer with neoadjuvant chemoradiation. There is a lack of consensus on how to handle the specimen, number of sections taken, correlation with pre- and post-operative radiological imaging, and especially, regression grading schema employed. Consensus in the form of guidelines is required so that the pathological assessment of these specimens will provide clinically relevant information for patient management, irrespective of location.

von Hippel–Lindau (VHL) disease is a hereditary autosomal dominant disorder associated with deletions or mutations in the VHL tumor suppressor gene. Characteristically, up to 60 % of neuroendocrine tumors (NETs) associated with VHL disease display a spectrum of clear cell morphology including multivacuolated lipid-rich cell change. Unlike neurofibromatosis type 1 and multiple endocrine neoplasia type 1 syndromes, ampullary NETs have not been described in association with VHL disease. In this report, we discuss the features of an incidental ampullary clear cell NET occurring in a patient with pancreatic VHL disease including multiple pancreatic NETs. The ampullary lesion consisted of epithelial cells resembling lipoblasts or signet ring cells. In our case, all NETs showing clear cell change were positive for inhibin. While the underlying mechanism of this finding is largely unknown, it is of note that positivity for inhibin has not been observed in clear cell NETs associated with multiple endocrine neoplasia type 1 syndrome. Our case proves that NETs can develop in the ampullary region in patients with VHL; clear cell change can occur in these lesions and can mimic signet ring cell carcinoma. This issue is of clinical significance especially in small biopsy samples; thus, positivity for keratin alone should not be taken as evidence of an adenocarcinoma. Moreover, demonstration of inhibin expression in a NET with clear cell change along with other clinical stigmata should alert the diagnostician to the possibility of VHL disease. However, further larger series examining inhibin expression in both syndrome-related and sporadic clear cell NETs are needed to confirm our findings.

A 46-year-old woman had a 4-month history of progressive cough, dysphagia, and dysphonia. Laryngoscopy revealed left vocal cord paralysis, and computed tomography demonstrated a mediastinal mass arising from the thyroid and invading the trachea and esophagus (Figure 1). A biopsy of the mass revealed papillary thyroid carcinoma (PTC). The patient underwent bilateral neck dissections and total thyroidectomy with en bloc resection of the mediastinal mass with 3 cm of trachea and outer muscular layer of the esophagus. Histological examination identified PTC with regions of poorly differentiated thyroid carcinoma (PDTC) in nodal metastases and in areas of extrathyroidal extension (pT4aN1b); margins were involved. Angioinvasion was characterized by intravascular tumor cells admixed with thrombus (1). Postsurgical thyroglobulin was undetectable ( 0.9 g/L); thyroglobulin antibodies were positive (183 IU/L). Following 200 mCi of radioactive iodine, no uptake outside the neck was identified. The patient received prophylactic external beam radiotherapy to the neck and superior mediastinum. Four months after surgery, she was found to have lung metastases with increasing thyroglobulin antibodies (551 IU/L). The low-burden lung disease remained relatively stable. Sixteen months after surgery, a postcricoid esophageal stricture was diagnosed. During gastroscopy to dilate it, a 1.5-cm suspicious nodule in the proximal body of the stomach was noted and biopsied. This was metastatic PTC with areas of PDTC. Although most thyroid carcinomas encountered in visceral organs represent metastatic disease, these can also arise within teratomas and ectopic thyroid tissues (2). Anaplastic thyroid carcinoma is known to metastasize to the stomach; however, intra-abdominal metastases from PTC or PDTC are extremely uncommon (3, 4). The application of rigid criteria to diagnose vascular invasion as identified in this case provides a clinically relevant prediction of distant metastasis in patients with thyroid carcinomas (1). To the best of our knowledge, this is the first reported gastric metastasis originating from PTC with regions of PDTC.

Introduction: Coeliac disease (CD) is an autoimmune condition characterised by gluten-induced intestinal inflammation, and observed at a 5-10 fold greater prevalence in type 1 diabetes. While universal screening for CD in patients with diabetes is frequently advocated, objective data is limited as to benefits on diabetes control, bone health or quality of life related to the adoption of a gluten-free diet (GFD) in the large proportion of patients with diabetes with asymptomatic CD. The Celiac Disease and Diabetes-Dietary Intervention and Evaluation Trial (CD-DIET) study is a multicenter, randomised controlled trial to evaluate the efficacy and safety of a GFD in patients with type 1 diabetes with asymptomatic CD. Methods and analysis: Children and adults (8-45 years) with type 1 diabetes will be screened for asymptomatic CD. Eligible patients with biopsy-proven CD will be randomly assigned in a 1:1 ratio to treatment with a GFD for 1 year, or continue with a gluten-containing diet. The primary outcome will evaluate the impact of the GFD on change in glycated haemoglobin. Secondary outcomes will evaluate changes in bone mineral density, blood glucose variability and health-related quality of life between GFD-treated and the regular diet group over a 1-year period. The study was initiated in 2012 and has subsequently expanded to multiple paediatric and adult centres in Ontario, Canada. Ethics and dissemination: The findings from this study will provide high-quality evidence as to the impact of GFD treatment on glycaemic control and complications in asymptomatic children and adults with CD and type 1 diabetes. Trial registration number: NCT01566110.

In this issue of the Journal of Clinical Pathology , MacGregor, Maughan and Sharma1 discuss the value and utility of pathological grading of regression from an oncologist's point of view. Neoadjuvant chemoradiotherapy is now a well-accepted treatment for locally advanced rectal cancer (cT3/T4 or lymph node-positive rectal cancers). However, neoadjuvant therapy affects the histopathological reporting of resected specimens by virtue of the host response to treatment, which occurs in the majority of patients. Pathological complete responses ranging from 9% to 29% have been reported,2 ,3 but the clinical significance of ‘incomplete’ regression and, more importantly, its role in determining postoperative treatment, is not clear. Pathological complete response has been associated with good patient outcomes.4–7 However, tumour regression grade (although having prognostic value for survival and recurrence by univariate analyses) has not been established to be an independent prognostic value that is superior to ypTNM in predicting clinical outcome.6 ,7–13 On the contrary, Min et al 11 demonstrated that regression grading is a good prognostic factor in patients with lymph node negative locally advanced rectal cancer. Therefore, regression grading may be a useful parameter for monitoring patient response and also as a potential prognostic factor. Several grading systems have been proposed and used, the most popular one being the Mandard and Dworak systems.14 ,15. We, an ‘International …

Tumor recurrence following treatment remains a major clinical challenge. Evidence from xenograft models and human trials indicates selective enrichment of cancer-initiating cells (C-ICs) in tumors that survive therapy. Together with recent reports showing that C-IC gene signatures influence patient survival, these studies predict that targeting self-renewal, the key stemness property unique to C-ICs, may represent a new paradigm in cancer therapy. Here we demonstrate that tumor formation, and more specifically human colorectal C-IC function are dependent on the canonical self-renewal regulator BMI-1. Down-regulation of BMI-1 inhibits the ability of colorectal C-ICs to self-renew resulting in the abrogation of their tumorigenic potential. Treatment of primary colorectal cancer xenografts with small molecule BMI-1 inhibitors resulted in colorectal C-IC loss with long-term and irreversible impairment of tumor growth. Targeting the Bmi-1 related self-renewal machinery provides the basis for a new therapeutic approach in the treatment of colorectal cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3496. doi:1538-7445.AM2012-3496