Colorectal cancer (CRC) is the fourth most frequently diagnosed cancer and the second leading cause of cancer death in the United States. Management of disseminated metastatic CRC involves various active drugs, either in combination or as single agents. The choice of therapy based on consideration of the goals of therapy, the type and timing of prior therapy, the mutational pro file of the tumor, and the differing toxicity pro files of the constituent drugs. This manuscript summarizes the data supporting the systemic therapy options recommended for metastatic CRC in the NCCN Guidelines for Colon Cancer.

The determination of an optimal treatment plan for an individual patient with rectal cancer is a complex process. In addition to decisions relating to the intent of rectal cancer surgery (ie, curative or palliative), consideration must also be given to the likely functional results of treatment, including the probability of maintaining or restoring normal bowel function/anal continence and preserving genitourinary functions. Particularly for patients with distal rectal cancer, finding a balance between curative-intent therapy while having minimal impact on quality of life can be challenging. Furthermore, the risk of pelvic recurrence is higher in patients with rectal cancer compared with those with colon cancer, and locally recurrent rectal cancer is associated with a poor prognosis. Careful patient selection and the use of sequenced multimodality therapy following a multidisciplinary approach is recommended. These NCCN Guidelines Insights detail recent updates to the NCCN Guidelines for Rectal Cancer, including the addition of endoscopic submucosal dissection as an option for early-stage rectal cancer, updates to the total neoadjuvant therapy approach based on the results of recent clinical trials, and the addition of a “watch-and-wait” nonoperative management approach for clinical complete responders to neoadjuvant therapy.

The determination of an optimal treatment plan for an individual patient with rectal cancer is a complex process. In addition to decisions relating to the intent of rectal cancer surgery (ie, curative or palliative), consideration must also be given to the likely functional results of treatment, including the probability of maintaining or restoring normal bowel function/anal continence and preserving genitourinary functions. Particularly for patients with distal rectal cancer, finding a balance between curative-intent therapy while having minimal impact on quality of life can be challenging. Furthermore, the risk of pelvic recurrence is higher in patients with rectal cancer compared with those with colon cancer, and locally recurrent rectal cancer is associated with a poor prognosis. Careful patient selection and the use of sequenced multimodality therapy following a multidisciplinary approach is recommended. These NCCN Guidelines Insights detail recent updates to the NCCN Guidelines for Rectal Cancer, including the addition of endoscopic submucosal dissection as an option for early- stage rectal cancer, updates to the total neoadjuvant therapy approach based on the results of recent clinical trials, and the addition of a "watch-and-wait" nonoperative management approach for clinical complete responders to neoadjuvant therapy.

Abstract Ulcerative colitis (UC) is a chronic inflammatory bowel disease (IBD) characterized by persistent mucosal inflammation extending throughout the colon. Due to compromised intestinal surface integrity and the prolonged use of medication, individuals with UC often develop a folate deficiency, requiring folic acid (FA) supplementation. Prior findings from this group indicate that FA supplementation, at clinically relevant levels, promotes UC-associated dysplasia. Supplementation with FA resulted in an increased multiplicity of dysplasia and a significant elevation in the expression of several inflammatory genes (e.g. IL-6, Cox-2, Iκκ-β) within the colonic mucosa of mice with UC. Genome-wide expression profiling of normal and dysplastic colonic epithelial cells from mice treated with different doses of FA revealed differentially expressed genes were enriched for members of the ERK and NF-κB pathways. Subsequent in vitro analyses, utilizing isogenic RKO colon adenocarcinoma cells exposed to various doses of FA, demonstrated the downregulation of ERK and p-ERK in wild type (WT) p53+/+ cells, while an increase in expression was observed in p53−/− cells at the protein level. The goal of this study is to investigate the role of the MAPK/ERK pathway in the development of UC-associated neoplasms. An in vitro model that faithfully reproduces the inflamed microenvironment in the context of WT vs. mutant p53, the putative gatekeeper of UC-associated carcinogenesis, is needed to enhance our understanding of early changes in the colon that are induced by FA and drive tumor formation. Colon organoids are being generated by isolating crypts from WT p53+/+ and mutant p53+/515A mice. These organoids are then co-cultured with mouse macrophages (RAW264.7 cells) in inserts to stimulate crosstalk between the macrophages and colonic epithelial cells, as in the inflamed colon. Analyses demonstrate that the macrophages express pivotal inflammatory cytokines characteristic of UC, including IL-1β, IL-10, TNF-α, and IL-6, irrespective of the presence or absence of LPS stimulation. Co-cultured organoids and macrophages will be exposed to different concentrations of FA for various lengths of time, and the impact of treatment on the levels of ERK, p-ERK, and p65 will be assessed. The ability of p-ERK to mediate the transcriptional activation of NF-κB and its downstream effector IL-6 will also be evaluated. Subsequent investigations employing this innovative experimental model will reveal the mechanisms by which FA supplementation promotes the progression of UC-associated dysplasia. Given the potential risks associated with FA supplementation in individuals with UC, there is an urgent need to translate these results to a clinical setting. (Supported by CA262551 and the Timothy and Aurora Hughes Cancer Research Fund). Citation Format: Ariane Rocha Bartolomeu, Wen-Chi Chang, Lisa Vanderveer, Kristen N. Harvey, Mitchell Cheung, Harry S. Cooper, Margie Lee Clapper. Effects of folic acid supplementation on early stages of colitis-associated carcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2170.

Breast cancer is treated with a multidisciplinary approach involving surgical oncology, radiation oncology, and medical oncology. The NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines) for Breast Cancer include recommendations for clinical management of patients with carcinoma in situ, invasive breast cancer, Paget's disease, Phyllodes tumor, inflammatory breast cancer, and management of breast cancer during pregnancy. The content featured in this issue focuses on the recommendations for overall management of systemic therapy (preoperative and adjuvant) options for nonmetastatic breast cancer. For the full version of the NCCN Guidelines for Breast Cancer, visit NCCN.org.

PURPOSE:The purpose of this study was to assess the effect of folic acid (FA) supplementation on colitis-associated colorectal cancer (CRC) using the azoxymethane/dextran sulfate sodium (AOM/DSS) model. METHODS:Mice were fed a chow containing 2 mg/kg FA at baseline and randomized after the first DSS treatment to receive 0, 2, or 8 mg/kg FA chow for 16 weeks. Colon tissue was collected for histopathological evaluation, genome-wide methylation analyses (Digital Restriction Enzyme Assay of Methylation), and gene expression profiling (RNA-Seq). RESULTS:A dose-dependent increase in the multiplicity of colonic dysplasias was observed, with the multiplicity of total and polypoid dysplasias higher (64% and 225%, respectively) in the 8 mg FA vs. the 0 mg FA group (p < 0.001). Polypoid dysplasias were hypomethylated, as compared to the non-neoplastic colonic mucosa (p < 0.05), irrespective of FA treatment. The colonic mucosa of the 8 mg FA group was markedly hypomethylated as compared to the 0 mg FA group. Differential methylation of genes involved in Wnt/β-catenin and MAPK signaling resulted in corresponding alterations in gene expression within the colonic mucosa. CONCLUSIONS:High-dose FA created an altered epigenetic field effect within the non-neoplastic colonic mucosa. The observed decrease in site-specific DNA methylation altered oncogenic pathways and promoted colitis-associated CRC.

ADT 061 pharmacokinetics and tissue distribution. ADT 061 was detected in plasma at a concentration of 2.8 µM 0.5 hr after a single oral administration of a dose of 100 mg/kg in 0.5% CMC/0.25% T80 in water to female C57BL/6 mice and reached plasma Cmax of 4.9 µM 4 hr after administration, which is >10 times higher than human colon cancer cell growth inhibition IC50 values. ADT 061 reached a Cmax in colon mucosa within 2 hr after oral administration (62.6 nmol/g) and was still present at concentrations exceeding IC50 values 8 hr after administration (28.9 nmol/g). ADT 061 concentrations in lungs, ovaries, and uterus (1-2 nmol/g) were appreciably lower than colon mucosa levels, while the ADT 061 concentration in the brain was nearly undetectable 2 and 8 hr post treatment (0.1 and 0.2 nmol/g, respectively) (n=4, mean {plus minus} SD).

HPLC chromatogram of compound 5.

HPLC chromatogram of ADT 061

Reaction steps for synthesis of ADT 061 (a) 3-(4-methoxyphenyl)-2-methylacrylic acid (compound 1): 4-Methoxybenzaldehyde (219 g, 1.61 mole), propionic anhydride (315g, 2.42 mole), and sodium propionate (155g, 1.61 mole) were stirred at 140 ºC until a clear solution was achieved (~48 h). The solution was cooled to room temperature and poured into 8L of ice water. The precipitation formed was collected by filtration, transferred into a 2L round-bottom flask, and refluxed in 1.5 L of ethanol for 3h. The flask was stored at -20 ºC overnight. Compound 1 was obtained as a colorless crystal (213g) after filtration. Compound 1 is also commercially available from AstraTech (# W18287,95% purity). (b) 3-(4-Methoxyphenyl)-2-methylpropanoic acid (compound 2): p-Methoxy-α-methylcinnamic acid (213g) and palladium on active charcoal (Pd-C, 10%, 2g) were suspended in 1.5 L of 95% ethanol and warmed to 60 ºC in a water bath. The warm suspension was immediately put on a catalytic hydrogenator, treated with hydrogen (40 psi). The reaction was completed within 45 min, as indicated by the complete dissolving of the solid starting material. The catalyst was removed by filtration, and the filtrate was concentrated to give compound 2 as a colorless oil (215g), which was used for the next reaction step without further purification and characterization. Compound 2 is also commercially available from PharmaBlocks (# PBTQ6955, 97% purity). (c) 6-Methoxy-2-methyl-2,3-dihydro-1H-inden-1-one (compound 3): Phosphoryl acid (98%, 500g) and polyphosphoryl acid (PPA, 450g) were pre-warmed separately to 70{degree sign}C in a water bath before being transferred to a 2L, three-necked flask equipped with a thermometer, a mechanical stirrer, and a dropping funnel. The flask was kept in an oil bath at 55-60 {degree sign}C for 1 h until smooth stirring was achieved. Compound 2 (95 g) was added dropwise over a period of 5 min. The temperature was carefully raised to 70-75{degree sign}C for 15 min. The reaction solution was immediately transferred to 6L of ice water and stirred until the PPA was completely dissolved. The mixture was extracted with ethyl ether (1L Ã- 3). The organic layer was dried with sodium sulfate and concentrated. The residue was purified by a silica gel column and eluted with hexane and acetone. Purity was monitored by TLC. Compound 3 was obtained as a clear, colorless oil (76g) that was used for the next reaction step without further purification and characterization. Compound 3 is also commercially available from AstraTech (# 96721, 95% purity). (d) 2-(5-Methoxy-2-methyl-1H-inden-3-yl) acetic acid (compound 4): A mixture of compound 3 (76g, 0.39 mole), cyanoacetic acid (36.6g, 0.43 mole), acetic acid (40 mL) and ammonium acetate (9 g) in 500 mL of toluene was refluxed with stirring for 48h. The liberated water was collected by a Dean-Stark trap. The reaction mixture was cooled and filtered, and the filtrate was concentrated. The residue was dissolved in 250 mL of ethanol. A solution of potassium hydroxide (90 g) in 360 mL of water was added to the solution. The mixture was refluxed overnight under argon. The organic solvents were removed under vacuum, and the remaining aqueous solution was diluted with 500 mL of water, extracted with ethyl ether, boiled with active charcoal for 1h, then filtered. The filtrate was acidified with 6N HCl and sonicated for 2h. The precipitate was collected by filtration, washed with water, refluxed in 180 mL of acetone for 2h, and stored at -20 ºC overnight. The precipitate was collected by filtration and dried under vacuum to give compound 4 as a colorless solid (42g). The mother liquor was concentrated, and a second crop (3.5g) was obtained after repeating the recrystallization procedure. The structure was confirmed by mass spectrometry (M+H: 219.04) and 1H-NMR (DMSO-d6) Î'(ppm): 7.240(d, 1H); 6.804(d, 1H); 6.658(m, 1H); 3.781 (s, 3H); 3.741(s, 2H); 3.250(s, 2H); 2.050(s, 3H). Purity was determined by HPLC (98.7%) (Suppl. Fig. 2a-c). (e) (Z)-2-(5-methoxy-2-methyl-1-(3,4,5-trimethoxybenzylidene)-1H-inden-3-yl)acetic acid (compound 5): Compound 4 (32.7g, 0.150 mole), 3, 4, 5-trimethoxybenzaldehyde (35.3g, 0.180 mole) and sodium methoxide (21g) in 300 mL of anhydrous methanol were refluxed in a 500 mL round-bottomed flask overnight. After cooling, the reaction mixture was diluted with 150 mL of acetone and sonicated for 30 min. The precipitate was collected by filtration and washed twice with acetone, then dissolved in 150 mL of water. The aqueous solution was acidified with 6N HCl and sonicated for 30 min. The precipitate was collected by filtration, washed with water, recrystallized from methanol, and dried under vacuum to afford compound 5 as a yellow crystal (51.7g). The structure was confirmed by mass spectrometry (M+H: 397.11) and 1H-NMR (DMSO-d6) Î'(ppm): 12.377 (s, 1H); 7.429 (d, 1H); 7.139 (s, 1H); 6.877(s, 2H); 6.669 (d, 1H); 6.546(dd, 1H); 3.780 (s, 3H); 3.780(s, 3H); 3.737(s, 6H); 3.545(s, 2H); 21.121(s, 3H). Purity was determined by HPLC (99.64%) (Suppl. Fig. 3a-c). (f) (Z)-2-(5-methoxy-2-methyl-1-(3,4,5-trimethoxybenzylidene)-1H-inden-3-yl)-N-(pyridin-3-yl) acetamide (ADT 061): To a 500 mL round-bottomed flask containing compound 5 (39.6g, 0.100 mole) and 300 mL of anhydrous dichloromethane, 1,1'-carbonyldiimidazole (CDI, 20.0g, 0.115 mole) was added in portions and stirred for 15 min at room temperature. 3-Amino-pyridine (10.6g, 0.115 mole) was added, followed by anhydrous pyridine (80 mL). The solution was stirred at 40{degree sign}C overnight, treated with 5g of sodium hydroxide in 20 mL of water for 10 min, then diluted with 300 mL of dichloromethane, and washed with water (250 mL x 3). The solution was concentrated, and the residue was purified with a silica gel column. Recrystallization from methanol and then from ethyl acetate afforded ADT 061 as a yellow crystal (32.7g). The structure of ADT 061 was confirmed by mass spectrometry (M+H: 473.2) and 1H-NMR (DMSO-d6) Î'(ppm): 10.453(sb, 1H); 8.765(d, 1H); 8.276(dd, 1H); 8.044(m, 1H); 7.431(d, 1H); 7.351(dd, 1H); 7.142(s,1H); 6.914(s, 1H); 6.880(s, 2H); 6.542(m, 1H); 3.781 (s, 6H); 3.731(s, 3H); 3.726(s, 3H); 3.696(s, 2H); 2.194(s, 3H). Purity was determined by HPLC (>99.7%) (Suppl. Fig. 4a-c).

Eadie-Hofstee plot of data from Fig. 2C. Error bars = SEM

This discussion summarizes the NCCN Clinical Practice Guidelines for managing squamous cell anal carcinoma, which represents the most common histologic form of the disease. A multidisciplinary approach including physicians from gastroenterology, medical oncology, surgical oncology, radiation oncology, and radiology is necessary. Primary treatment of perianal cancer and anal canal cancer are similar and include chemoradiation in most cases. Follow-up clinical evaluations are recommended for all patients with anal carcinoma because additional curative-intent treatment is possible. Biopsy-proven evidence of locally recurrent or persistent disease after primary treatment may require surgical treatment. Systemic therapy is generally recommended for extrapelvic metastatic disease. Recent updates to the NCCN Guidelines for Anal Carcinoma include staging classification updates based on the 9th edition of the AJCC Staging System and updates to the systemic therapy recommendations based on new data that better define optimal treatment of patients with metastatic anal carcinoma.

Supplemental Figure Legends

Supplementary Data 1-13 from One-Hit Effects in Cancer: Altered Proteome of Morphologically Normal Colon Crypts in Familial Adenomatous Polyposis

AST and ALT activity of male APC+/Min-FCCC mice administered varying doses of ADT 061 in the diet. Enzyme activity was measured in plasma samples. Results are presented as mean {plus minus} SEM.

Mass spectroscopy of compound 4.

Detection of colorectal dysplasia during surveillance colonoscopy remains the best method of determining risk for colitis-associated colorectal cancer (CAC). miRNAs (miRs) show great promise as tissue-specific biomarkers of neoplasia. The goal of this study was to explore the miR expression profile of precancerous dysplastic lesions in the AOM/DSS mouse model and identify early molecular changes associated with CAC. Epithelial cells were laser-microdissected from the colonic mucosa (inflamed versus dysplastic) of mice with AOM/DSS-induced colitis. A miR signature that can distinguish inflamed non-neoplastic mucosa from dysplasia was identified. Bioinformatic analyses led to the discovery of associated miR gene targets and enriched pathways and supported the construction of a network interaction map. miR-1a-3p was one of the miRs with the highest number of predicted targets, including Cdk6. Interestingly, miR-1a-3p and Cdk6 were down- and up-regulated in dysplastic lesions, respectively. Transfection of HCT116 and RKO cells with miR-1a-3p mimics induced apoptosis and cell cycle arrest in G1, suggesting its biological function. A slight reduction in the level of CDK6 transcripts was also observed in cells transfected with miR-1. These data provide novel insight into the early molecular alterations that accompany the development of CAC and identify a miR signature that represents a promising biomarker for the early detection of colitis-associated dysplasia.

Many studies show significantly improved survival after R0 resection compared with R1 resection in pancreatic adenocarcinoma (PAC); however, the effect of neoadjuvant chemoradiation (NACRT) on this association is unknown. The aim of this study was to evaluate the prognostic significance of positive surgical margins (SMs) after NACRT compared with upfront surgery + adjuvant therapy in PAC. All cases of surgically resected PAC at a single institution were reviewed from 1996 to 2014; patients treated with palliative intent, metastatic disease, and biliary/ampullary tumors were excluded. The primary endpoint was overall survival (OS). Overall, 300 patients were included; 134 patients received NACRT with concurrent 5-fluorouracil or gemcitabine followed by surgery, and 166 patients received upfront surgery (+ adjuvant chemotherapy in 72% of patients and RT in 65%); 31% of both groups had a positive SM (+SM). The median OS for patients with a +SM or negative SM (−SM) was 26.6 and 31.6 months, respectively for NACRT, and 12.0 and 24.5 months, respectively, for upfront surgery. OS was significantly improved with −SM compared with +SM in both groups (p = 0.006). When resection yielded +SM, NACRT patients had improved OS compared with upfront surgery patients (p < 0.001). On multivariable analysis, +SM in the upfront surgery group (hazard ratio [HR] 2.94, 95% confidence interval [CI] 2.04–4.24; p < 0.001) and older age (HR 1.01, 95% CI 1.00–1.03, per year; p = 0.007) predicted worse OS. +SM in the NACRT group was not associated with worse OS (HR 1.09, 95% CI 0.72–1.65; p = 0.70). Patients with a positive margin after NACRT and surgery had longer survival compared with patients with a positive margin after upfront surgery. NACRT should be strongly considered for patients at high risk of R1 resections.

This selection from the NCCN Guidelines for Rectal Cancer focuses on management of malignant polyps and resectable nonmetastatic rectal cancer because important updates have been made to these guidelines. These recent updates include redrawing the algorithms for stage II and III disease to reflect new data supporting the increasingly prominent role of total neoadjuvant therapy, expanded recommendations for short-course radiation therapy techniques, and new recommendations for a "watch-and-wait" nonoperative management technique for patients with cancer that shows a complete response to neoadjuvant therapy. The complete version of the NCCN Guidelines for Rectal Cancer, available online at NCCN.org, covers additional topics including risk assessment, pathology and staging, management of metastatic disease, posttreatment surveillance, treatment of recurrent disease, and survivorship.

Detection of colorectal dysplasia during surveillance colonoscopy is currently the best method of determining risk of colitis-associated colorectal cancer (CAC). An understanding of the early molecular changes associated with the development of these lesions will inform the identification of new biomarkers for earlier detection. miRNAs (miRs), highly conserved noncoding RNAs, show great promise as stable, tissue-specific biomarkers of neoplasia. We previously identified 12 miRs that are differentially-expressed in colitis-associated dysplasias (flat and polypoid) vs. inflamed colonic mucosa from mice treated with AOM/DSS. miR-1, a putative tumor suppressor, was downregulated in colitis-associated dysplasias. Analysis of the mRNA expression profile of AOM/DSS-induced dysplasias and prediction of the interactions between the miRs and their targets led to the selection of the Cdk6 as the target of miR-1 to be further investigated, based on its: 1) upregulation in AOM/DSS-induced dysplasia; and 2) association with cell cycle progression and inflammatory signaling. The goal of the present study was to validate the predicted interaction between miR-1 and Cdk6 and assess the biological function of miR-1 in vitro. The ability of miR-1 to interact with the 3’UTR of Cdk6 mRNA was assessed using a dual luciferase assay. Co-transfection of HCT116 or RKO colon carcinoma cells with Cdk6-WT and miR-1 mimics led to a significant reduction in relative luciferase activity in both cell lines (30%, p=0.0117 and 47%, p=0.0269; respectively). Transfection with the Cdk6-Mut did not alter relative luciferase activity, confirming the Cdk6 binding site was specific for miR-1. The biological function of miR-1 was assessed in HCT116 and RKO cells reverse-transfected with miR-1 and cel-miR-67 (negative control) for 48 hrs. Apoptosis (AnnexinV+ cells) and cell cycle progression (% of cells in G0/G1, G2/M and S phase) were evaluated by flow cytometry, and proliferation by cell count (Trypan Blue). HCT116 and RKO transfected with the miR-1 mimic exhibited a higher proportion of apoptotic cells than the negative control (30% and 20% increase, p=0.0022 and 0.0013, respectively). Cell cycle analyses revealed the miR-1 mimic induced cell cycle arrest (G0/G1) in both cell lines (p<0.05). In HCT116 cells, this was accompanied by a reduction in the percentage of cells in G2/M (p=0.002) and S (p<0.001) phase. No effect of miR-1 on total cell number was observed. These results demonstrate that Cdk6 is a direct target of miR-1, and suggest that downregulation of this miR in dysplastic lesions contributes to CAC by inducing cell cycle progression and inhibiting apoptosis. These data provide novel insight into the early molecular changes that accompany the development of colitis-associated dysplasia and may serve as biomarkers for early detection of neoplasia. Supported by the Timothy P. and Aurora M. Hughes Fund for Colon Cancer Research. Citation Format: Mariana F. Fragoso, Geysson J. Fernandez, Lisa Vanderveer, Harry S. Cooper, Michael Slifker, Margie L. Clapper. miR-1 targets Cdk6 and controls cell cycle progression and apoptosis in colitis-associated dysplasia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1553.