Clinical variables associated with immune checkpoint inhibitor outcomes in patients with metastatic urothelial carcinoma: a multicentre retrospective cohort study.

OBJECTIVES: Immune checkpoint inhibitors (ICIs) are indicated for metastatic urothelial cancer (mUC), but predictive and prognostic factors are lacking. We investigated clinical variables associated with ICI outcomes. METHODS: We performed a multicentre retrospective cohort study of 135 patients who received ICI for mUC, 2016-2021, at three Canadian centres. Clinical characteristics, body mass index (BMI), metastatic sites, neutrophil-to-lymphocyte ratio (NLR), response and survival were abstracted from chart review. RESULTS: We identified 135 patients and 62% had received ICI as a second-line or later treatment for mUC. A BMI ≥25 was significantly correlated to a higher overall response rate (ORR) (45.4% vs 16.3%, p value=0.020). Patients with BMI ≥30 experienced longer median overall survival (OS) of 24.8 vs 14.4 for 25≤BMI<30 and 8.5 months for BMI <25 (p value=0.012). The ORR was lower in the presence of bone metastases (16% vs 41%, p value=0.006) and liver metastases (16% vs 39%, p value=0.013). Metastatic lymph nodes were correlated with higher ORR (40% vs 20%, p value=0.032). The median OS for bone metastases was 7.3 versus 18 months (p value <0.001). Patients with liver metastases had a median OS of 8.6 versus 15 months (p value=0.006). No difference for lymph nodes metastases (13.5 vs 12.7 months, p value=0.175) was found. NLR ≥4 had worse OS (8.2 vs 17.7 months, p value=0.0001). In multivariate analysis, BMI ≥30, bone metastases, NLR ≥4, performance status ≥2 and line of ICI ≥2 were independent factors for OS. CONCLUSIONS: Our data identified BMI and bone metastases as novel clinical biomarkers that were independently associated with ICI outcomes in mUC. External and prospective validation are warranted.

Statins to prevent early cardiac dysfunction in cancer patients at increased cardiotoxicity risk receiving anthracyclines.

BACKGROUND AND AIMS: Anthracyclines can cause cancer therapy-related cardiac dysfunction (CTRCD). We aimed to assess whether statins prevent decline in left ventricular ejection fraction (LVEF) in anthracycline-treated patients at increased risk for CTRCD. METHODS: In this multicenter double-blinded, placebo-controlled trial, patients with cancer at increased risk of anthracycline-related CTRCD (per ASCO guidelines) were randomly assigned to atorvastatin 40 mg or placebo once-daily. Cardiovascular magnetic resonance (CMR) imaging was performed before and within 4 weeks after anthracyclines. Blood biomarkers were measured at every cycle. The primary outcome was post-anthracycline LVEF, adjusted for baseline. CTRCD was defined as a fall in LVEF by >10% to <53%. Secondary endpoints included left ventricular (LV) volumes, CTRCD, CMR tissue characterization, high sensitivity troponin I (hsTnI), and B-type natriuretic peptide (BNP). RESULTS: We randomized 112 patients (56.9 ± 13.6 years, 87 female, and 73 with breast cancer): 54 to atorvastatin and 58 to placebo. Post-anthracycline CMR was performed 22 (13-27) days from last anthracycline dose. Post-anthracycline LVEF did not differ between the atorvastatin and placebo groups (57.3 ± 5.8% and 55.9 ± 7.4%, respectively) when adjusted for baseline LVEF (P = 0.34). There were no significant between-group differences in post-anthracycline LV end-diastolic (P = 0.20) or end-systolic volume (P = 0.12), CMR myocardial edema and/or fibrosis (P = 0.06-0.47), or peak hsTnI (P ≥ 0.99) and BNP (P = 0.23). CTRCD incidence was similar (4% versus 4%, P ≥ 0.99). There was no difference in adverse events. CONCLUSIONS: In patients at increased risk of CTRCD, primary prevention with atorvastatin during anthracycline therapy did not ameliorate early LVEF decline, LV remodeling, CTRCD, change in serum cardiac biomarkers, or CMR myocardial tissue changes. TRIAL REGISTRATION: NCT03186404.

Key Considerations for the Treatment of Advanced Breast Cancer in Older Adults: An Expert Consensus of the Canadian Treatment Landscape.

The prevalence of breast cancer amongst older adults in Canada is increasing. This patient population faces unique challenges in the management of breast cancer, as older adults often have distinct biological, psychosocial, and treatment-related considerations. This paper presents an expert consensus of the Canadian treatment landscape, focusing on key considerations for optimizing selection of systemic therapy for advanced breast cancer in older adults. This paper aims to provide evidence-based recommendations and practical guidance for healthcare professionals involved in the care of older adults with breast cancer. By recognizing and addressing the specific needs of older adults, healthcare providers can optimize treatment outcomes and improve the overall quality of care for this population.

Impact of body mass index on survival in women receiving chemotherapy for early breast cancer.

PURPOSE: The impact of elevated body mass index (BMI) on overall survival (OS) in patients receiving modern anthracycline-taxane chemotherapy for early breast cancer (EBC) has not yet been well established. The purpose of our study was to examine overall survival (OS) by BMI category in women with EBC receiving either doxorubicin (A), cyclophosphamide (C) + paclitaxel (P) or fluorouracil (F), epirubicin (E), cyclophosphamide (C) + docetaxel (D). METHODS: This was a retrospective cohort study in patients ≥ 18 years with resected stage I-III BC diagnosed between 2007 and 2017 in Ontario, identified through linkage of administrative databases. Patients were classified according to baseline BMI into underweight (< 18.5 kg/m2), normal (18.5-24.9 kg/m2), overweight (25-29.9 kg/m2), and obese (≥ 30 kg/m2) World Health Organization (WHO) categories. The primary outcome was OS. Univariable and multivariable analyses were used to examine the association between clinico-pathologic characteristics and OS among BMI categories. RESULTS: Our cohort included 11,601 women, of whom 3890 (33.5%) were normal weight, 3696 (31.9%) overweight, and 3847 (33.1%) obese. Median OS was 7.9 years. There were no statistically significant differences in OS according to BMI (p = 0.66) in the overall study cohort or among the BMI categories after adjusting for age, nodal status, stage, grade, ER and HER2 status for either AC-P or FEC-D- treated patients (p = 0.45 and p = 0.97, respectively). CONCLUSIONS: Our large population-based retrospective cohort analysis of EBC patients receiving adjuvant anthracycline-taxane chemotherapy found no significant impact of BMI on OS. Further investigation is warranted to confirm these findings in prospective patient cohorts.

Helicobacter pylori serology is associated with worse overall survival in patients with melanoma treated with immune checkpoint inhibitors.

The microbiome is now regarded as one of the hallmarks of cancer and several strategies to modify the gut microbiota to improve immune checkpoint inhibitor (ICI) activity are being evaluated in clinical trials. Preliminary data regarding the upper gastro-intestinal microbiota indicated that Helicobacter pylori seropositivity was associated with a negative prognosis in patients amenable to ICI. In 97 patients with advanced melanoma treated with ICI, we assessed the impact of H. pylori on outcomes and microbiome composition. We performed H. pylori serology and profiled the fecal microbiome with metagenomics sequencing. Among the 97 patients, 22% were H. pylori positive (Pos). H. pylori Pos patients had a significantly shorter overall survival (p = .02) compared to H. pylori negative (Neg) patients. In addition, objective response rate and progression-free survival were decreased in H. pylori Pos patients. Metagenomics sequencing did not reveal any difference in diversity indexes between the H. pylori groups. At the taxa level, Eubacterium ventriosum, Mediterraneibacter (Ruminococcus) torques, and Dorea formicigenerans were increased in the H. pylori Pos group, while Alistipes finegoldii, Hungatella hathewayi and Blautia producta were over-represented in the H. pylori Neg group. In a second independent cohort of patients with NSCLC, diversity indexes were similar in both groups and Bacteroides xylanisolvens was increased in H. pylori Neg patients. Our results demonstrated that the negative impact of H. pylori on outcomes seem to be independent from the fecal microbiome composition. These findings warrant further validation and development of therapeutic strategies to eradicate H. pylori in immuno-oncology arena.

FAM72A antagonizes UNG2 to promote mutagenic repair during antibody maturation.

Activation-induced cytidine deaminase (AID) catalyses the deamination of deoxycytidines to deoxyuracils within immunoglobulin genes to induce somatic hypermutation and class-switch recombination1,2. AID-generated deoxyuracils are recognized and processed by subverted base-excision and mismatch repair pathways that ensure a mutagenic outcome in B cells3-6. However, why these DNA repair pathways do not accurately repair AID-induced lesions remains unknown. Here, using a genome-wide CRISPR screen, we show that FAM72A is a major determinant for the error-prone processing of deoxyuracils. Fam72a-deficient CH12F3-2 B cells and primary B cells from Fam72a-/- mice exhibit reduced class-switch recombination and somatic hypermutation frequencies at immunoglobulin and Bcl6 genes, and reduced genome-wide deoxyuracils. The somatic hypermutation spectrum in B cells from Fam72a-/- mice is opposite to that observed in mice deficient in uracil DNA glycosylase 2 (UNG2)7, which suggests that UNG2 is hyperactive in FAM72A-deficient cells. Indeed, FAM72A binds to UNG2, resulting in reduced levels of UNG2 protein in the G1 phase of the cell cycle, coinciding with peak AID activity. FAM72A therefore causes U·G mispairs to persist into S phase, leading to error-prone processing by mismatch repair. By disabling the DNA repair pathways that normally efficiently remove deoxyuracils from DNA, FAM72A enables AID to exert its full effects on antibody maturation. This work has implications in cancer, as the overexpression of FAM72A that is observed in many cancers8 could promote mutagenesis.

MR-guided focused ultrasound enhances delivery of trastuzumab to Her2-positive brain metastases.

The blood-brain barrier (BBB) is an important factor limiting the effectiveness of central nervous system (CNS) therapeutics. MR-guided focused ultrasound (MRgFUS) is a noninvasive, spatially precise technology that enhances drug delivery across a temporarily permeable BBB. However, despite promising preclinical data, successful drug delivery has yet to be proven in human patients. In this study, we provide primary evidence of enhanced brain penetration of trastuzumab with MRgFUS in patients with Her2-positive breast cancer and brain metastases (NCT03714243). Four patients with progressive intracranial disease and stable systemic disease were enrolled in a single-arm open-labeled study. Twenty treatments combining transcranial MRgFUS with concomitant standard-of-care intravenous trastuzumab-based therapies were administered as outpatient procedures. The primary outcome was safety, and there were no treatment-related serious adverse events. The efficacy of trastuzumab delivery was demonstrated using 111In-BzDTPA-NLS-trastuzumab SPECT imaging. The standardized uptake value ratio (SUVR) of MRgFUS-treated lesions increased, on average, by 101 ± 71%, compared to −18 ± 26% in control lesions. MRgFUS enhanced drug uptake in 87 ± 17% of sonicated voxels (>20% increase in SUVR), with up to a 450% voxel-wise increase detected. Control lesions had 8 ± 8% voxels with >20% increase in SUVR. With treatment, unidimensional tumor measurements decreased by 19 ± 12%. This study provides first-in-human evidence of noninvasive, spatially targeted monoclonal antibody delivery across the BBB using MRgFUS, demonstrating the promise of this technology for a broad range of CNS diseases.

Association of Antibiotics and Other Drugs with Clinical Outcomes in Metastatic Melanoma Patients Treated with Immunotherapy.

Immune checkpoint inhibitors (ICIs) targeting the programmed cell death protein-1 (PD-1) and programmed cell death ligand-1 (PD-L1) have improved survival in many advanced cancers including advanced melanoma, renal cell, urothelial, and non-small-cell lung cancers. However, not all patients respond, and immune-related adverse events (irAEs) are common. Commensal gut bacteria may serve as an immunoregulatory link-mediating ICI response and toxicity. Recent studies have shown that a lack of bacterial diversity, known as gut dysbiosis, can have an adverse impact on patients' response to ICIs and predispose to the development of irAEs. Data were collected from 167 patients with metastatic melanoma who received antibiotics within 30 days prior to and/or after initiation of ICI and patients who received NSAIDs, statins, steroids, or proton-pump inhibitors (PPI) within 30 days prior to ICI initiation. The primary outcome was time-to-discontinuation (TTD) of ICI therapy, measured from the date of ICI initiation to the last treatment date. The secondary outcome of interest was toxicity, with incidence of irAEs graded as per the Common Terminology Criteria for Adverse Events (CTCAE), version 5.0. Here, we demonstrate that individuals who received antibiotics had a significantly shorter time-to-discontinuation (TTD) of the ICI therapy as opposed those who were not administered antibiotics. Consistent with results from previous research, we propose that antibiotics have a negative effect on a patient's response to ICI therapy, most likely due to the result of gut dysbiosis, and should be critically assessed in terms of their use in patients undergoing ICI treatment.

Gut microbiota signatures are associated with toxicity to combined CTLA-4 and PD-1 blockade.

Treatment with combined immune checkpoint blockade (CICB) targeting CTLA-4 and PD-1 is associated with clinical benefit across tumor types, but also a high rate of immune-related adverse events. Insights into biomarkers and mechanisms of response and toxicity to CICB are needed. To address this, we profiled the blood, tumor and gut microbiome of 77 patients with advanced melanoma treated with CICB, with a high rate of any ≥grade 3 immune-related adverse events (49%) with parallel studies in pre-clinical models. Tumor-associated immune and genomic biomarkers of response to CICB were similar to those identified for ICB monotherapy, and toxicity from CICB was associated with a more diverse peripheral T-cell repertoire. Profiling of gut microbiota demonstrated a significantly higher abundance of Bacteroides intestinalis in patients with toxicity, with upregulation of mucosal IL-1ß in patient samples of colitis and in pre-clinical models. Together, these data offer potential new therapeutic angles for targeting toxicity to CICB.

Patients' and Oncologists' Knowledge and Expectations Regarding Tumor Multigene Next-Generation Sequencing: A Narrative Review.

BACKGROUND: Tumor multigene next-generation sequencing (NGS) is increasingly being offered to cancer patients to guide clinical management and determine eligibility for clinical trials. We undertook a review of studies examining the knowledge and attitudes of patients and oncologists regarding the primary results and potential secondary findings of such testing. MATERIALS AND METHODS: A search was conducted through the MEDLINE database using the following keywords: "neoplasms" and "molecular sequencing / genome sequencing / tumor profiling / NGS / whole exome sequencing" and "patient / oncologist" and "knowledge / attitudes / satisfaction / experience / evaluation / perspective / practice / preference." Articles meeting the inclusion criteria and additional relevant articles from their references were selected. RESULTS: From 1,142 publications identified by the search and 9 from references, 21 publications were included in the final review. Patients generally had positive attitudes toward tumor NGS despite relatively little knowledge of test-related genetics concepts, but their expectations often exceeded the reality of low clinical utility. Patients with higher education and greater genetics knowledge had more realistic expectations and a more altruistic view of the role of NGS. Attitudes toward disclosure of secondary findings were highly variable. Oncologists had poor to moderate genomic literacy; they communicated challenges with tempering patient expectations and deciding what information to disclose. CONCLUSION: Patients considering undergoing tumor NGS should be provided with easily understandable resources explaining the procedure, goals, and probable outcomes, whenever possible based on evidence-based guidelines. Continuing medical education programs on this topic for oncology health care professionals should strive to improve their genomic literacy and instruct them on how to optimally present this information to their patients. IMPLICATIONS FOR PRACTICE: Oncologists are increasingly offering tumor multigene testing to patients with advanced cancers to guide more "personalized" treatment and/or determine eligibility for clinical trials. However, patients often have inadequate understanding and unrealistic expectations. Oncologists must ensure that they themselves have sufficient knowledge of the benefits and limitations of testing and must provide their patients with appropriate educational resources. Prior to testing, patients should be told the likelihood of finding a mutation in their specific tumor type for which a targeted treatment or clinical trial is available. Patients also need clear information about the possibility and implications of secondary findings.

Impact of obesity on clinical outcomes in hormone receptor-positive breast cancer: a systematic review.

BACKGROUND: The relationship between obesity and prognosis of early breast cancer is complex. Increased levels of aromatase present in adipose tissue of obese postmenopausal women may lead to suboptimal suppression of systemic estrogens. However, studies have been mixed with respect to the association between use of aromatase inhibitors (AIs) and clinical outcomes in obese women with early breast cancer. METHODS: We conducted a systematic literature review following PRISMA guidelines to examine the impact of obesity on the efficacy of AIs in early-stage hormone receptor-positive breast cancer. Primary outcome measures included disease-free survival, relapse-free survival, distant recurrence-free survival, breast cancer-free survival, and overall survival. RESULTS: Of 491 studies identified, eight studies met criteria for inclusion: three retrospective cohort studies, one prospective cohort study and four randomized controlled trials. Four studies limited eligibility to postmenopausal women. Percentage of obese patients in studies ranged from 10 to 30%. Two studies examined use of AIs alone while the remainder included patients treated with either AIs or tamoxifen. Five out of seven studies suggested a negative impact of obesity on AI efficacy. CONCLUSIONS: The results of our systematic review highlight a need for further research exploring the optimal endocrine therapies for obese women. There is insufficient evidence at present to recommend tailoring adjuvant endocrine therapy with use of specific AIs or for dosing modifications of AIs in this patient population.

Quality of adverse event reporting in phase III randomized controlled trials of breast and colorectal cancer: A systematic review.

BACKGROUND: Clinical trial reports often emphasize efficacy over harms, leading to misinterpretation of the risk-to-benefit ratio of new therapies. Clear and sufficiently detailed reporting of methods and results is especially important in the abstracts of trial reports, as readers often base their assessment of a trial on such information. In this study, we evaluated the quality of adverse event (AE) reporting and abstract quality in phase III randomized controlled trials (RCTs) of systemic therapies in breast and colorectal cancer. METHODS: Medline, EMBASE, Cochrane Database of RCTs, and Cochrane Database of Systematic Reviews were searched from November 2005 to September 2018. Phase III RCTs evaluating systemic therapies in breast or colorectal cancer were included. Each article was independently reviewed by two investigators using a standardized data extraction form based on guidelines developed by the Consolidated Standards of Reporting Trials (CONSORT) group. Descriptive statistics, bivariate analysis, and multivariable linear regression were used to analyze data. All statistical tests were two-sided. RESULTS: Of 166 RCTs identified, 99.4% reported harms in the manuscript body, and 59.6% reported harms in the abstract. Reporting was restricted to severe harms in 15.6% of RCTs. Statistical comparison of AE rates went unreported in 59.0% of studies. Information regarding AEs leading to dose reductions, treatment discontinuations, or study withdrawals went unreported in 59.3%, 18.7%, and 86.8% of studies, respectively. Recently published RCTs (P = .009) and those sponsored at least partially by for-profit companies (P = .003) had higher abstract quality scores. CONCLUSIONS: Breast and colorectal cancer phase III RCTs inadequately report CONSORT-compliant AE data. Improved guideline adherence and abstract reporting is required to properly weigh benefits and harms of new oncologic therapies. SYSTEMATIC REVIEW REGISTRATION NUMBER: CRD42019140673.

Impact of the gut microbiota on immune checkpoint inhibitor-associated toxicities.

Immune checkpoint inhibitors (ICIs) have transformed the treatment of patients with advanced cancers. However, the majority of patients do not respond or develop early progressive disease. A substantial number also develop immune-mediated toxicities that may lead to early treatment discontinuation. Gastrointestinal toxicities in the form of diarrhea and colitis are common and may resemble that observed in patients with inflammatory bowel disease (IBD). Alterations in the gut microbiota are thought to play an important role in mediating the intestinal inflammation that is associated with immune-mediated colitis. In this review, the authors' objective is to provide an overview of the gastrointestinal and hepatic toxicities that can be seen with ICIs and discuss the interactions between gut microbiota and the immune response. The authors also highlight the potential role for fecal microbial transfer (FMT) as an approach to improve therapeutic efficacy and decrease toxicity.

Underuse of ECG monitoring in oncology patients receiving QT-interval prolonging drugs.

OBJECTIVE: We examined use of ECG monitoring in oncology patients prescribed QT-prolonging drugs. METHODS: Patients ≥66 years diagnosed with cancer between 2005 and 2011 were identified through the Ontario Cancer Registry and linked to multiple population-based administrative databases to ascertain demographics, comorbidities, prescription drug use, systemic therapy and ECG. QT-prolonging drugs were identified as per drug lists developed by the Arizona Center for Education and Research on Therapeutics. Univariable and multivariable analyses were used to examine factors associated with ECG use in patients on first-line systemic therapy. RESULTS: A total of 48 236 patients (median age 74; 49% women) received one or more drugs associated with a risk of QT-interval prolongation but only 27% of patients had an ECG performed. Factors associated with more ECG use on multivariable analysis included recent cancer diagnosis (p for trend <0.001 between 2005 and 2011), use of concurrent QT-prolonging drugs (OR=1.15 per each additional QT-prolonging drug, 95% CI 1.12 to 1.17) and the presence of coronary artery disease (OR 1.31; 95% CI 1.25 to 1.38) and heart failure (OR 1.25; 95% CI 1.17 to 1.35). Use of anticancer (OR 0.74; 95% CI 0.70 to 0.79) and antiemetic (OR 0.93; 95% CI 0.88 to 0.99) QT-prolonging drugs was paradoxically associated with less ECG use. CONCLUSIONS: Our study highlights common use of QT-prolonging drugs and underuse of ECG in oncology patients. Since ECG is an inexpensive, non-invasive and widely available test, it may be readily incorporated in the monitoring of patients for toxicities in routine clinical practice.

Impact of multi-gene mutational profiling on clinical trial outcomes in metastatic breast cancer.

PURPOSE: Next-generation sequencing (NGS) has identified recurrent genomic alterations in metastatic breast cancer (MBC); however, the clinical utility of incorporating routine sequencing to guide treatment decisions in this setting is unclear. We examine the frequency of genomic alterations in MBC patients from academic and community hospitals and correlate with clinical outcomes. METHODS: MBC patients with good performance status were prospectively recruited at the Princess Margaret Cancer Centre (PM) in Canada. Molecular profiling on DNA extracted from FFPE archival tissues was performed on the Sequenom MassArray platform or the TruSeq Amplicon Cancer Panel (TSACP) on the MiSeq platform. Clinical trial outcomes by RECIST 1.1 and time on treatment were reviewed retrospectively. RESULTS: From January 2012 to November 2015, 483 MBC patients were enrolled and 440 were genotyped. At least one somatic mutation was identified in 46% of patients, most commonly in PIK3CA (28%) or TP53 (13%). Of 203 patients with ≥ 1 mutation(s), 15% were treated on genotype-matched and 9% on non-matched trials. There was no significant difference for median time on treatment for patients treated on matched vs. non-matched therapies (3.6 vs. 3.8 months; p = 0.89). CONCLUSIONS: This study provides real-world outcomes on hotspot genotyping and small targeted panel sequencing of MBC patients from academic and community settings. Few patients were matched to clinical trials with targeted therapies. More comprehensive profiling and improved access to clinical trials may increase therapeutic options for patients with actionable mutations. Further studies are needed to evaluate if this approach leads to improved clinical outcomes.

Noncoding somatic and inherited single-nucleotide variants converge to promote ESR1 expression in breast cancer.

Sustained expression of the estrogen receptor-α (ESR1) drives two-thirds of breast cancer and defines the ESR1-positive subtype. ESR1 engages enhancers upon estrogen stimulation to establish an oncogenic expression program. Somatic copy number alterations involving the ESR1 gene occur in approximately 1% of ESR1-positive breast cancers, suggesting that other mechanisms underlie the persistent expression of ESR1. We report significant enrichment of somatic mutations within the set of regulatory elements (SRE) regulating ESR1 in 7% of ESR1-positive breast cancers. These mutations regulate ESR1 expression by modulating transcription factor binding to the DNA. The SRE includes a recurrently mutated enhancer whose activity is also affected by rs9383590, a functional inherited single-nucleotide variant (SNV) that accounts for several breast cancer risk-associated loci. Our work highlights the importance of considering the combinatorial activity of regulatory elements as a single unit to delineate the impact of noncoding genetic alterations on single genes in cancer.

Gut microbial metabolism drives transformation of MSH2-deficient colon epithelial cells.

The etiology of colorectal cancer (CRC) has been linked to deficiencies in mismatch repair and adenomatous polyposis coli (APC) proteins, diet, inflammatory processes, and gut microbiota. However, the mechanism through which the microbiota synergizes with these etiologic factors to promote CRC is not clear. We report that altering the microbiota composition reduces CRC in APC(Min/+)MSH2(-/-) mice, and that a diet reduced in carbohydrates phenocopies this effect. Gut microbes did not induce CRC in these mice through an inflammatory response or the production of DNA mutagens but rather by providing carbohydrate-derived metabolites such as butyrate that fuel hyperproliferation of MSH2(-/-) colon epithelial cells. Further, we provide evidence that the mismatch repair pathway has a role in regulating ß-catenin activity and modulating the differentiation of transit-amplifying cells in the colon. These data thereby provide an explanation for the interaction between microbiota, diet, and mismatch repair deficiency in CRC induction. PAPERCLIP:

Altered dynamics of intestinal cell maturation in Apc1638N/+ mice.

Novel imaging of active transcription sites in interphase nuclei of intestinal epithelial cells in situ showed that key genes associated with Wnt and Notch signaling were dynamically regulated as the cells underwent normal maturation during their migration along the mouse crypt-villus axis (CVA). However, oscillating patterns of activation of these genes were displaced along this axis in the histologically normal intestinal mucosa of Apc(1638N/+) mice before tumor development. Gene expression profiling then showed that the normal reprogramming of cells along the CVA was dampened in the Apc(1638N/+) mice, with an overrepresentation of c-myc target genes among those loci affected in the mutant mice. Moreover, in the Apc(1638N/+) mice, there was a perturbed pattern of expression of lineage-specific markers along the CVA consistent with transcription site repression of the Math1 gene, and genes encoding enzymes of every step of the tricarboxylic acid cycle were downregulated in the crypt of Apc(1638N/+) mice compared with WT, but not in the villus. These changes may alter energy metabolism and generate a pseudohypoxic state, suggested by elevated expression of Hif1alpha and its target genes. Thus, although intestinal tumors develop in Apc(1638N/+) mice on focal loss or inactivation of the WT allele, our results show that in the Apc(1638N/+) mouse, inheritance of only a single WT Apc allele perturbs the dynamic and complex reprogramming underlying normal cell maturation, which links epithelial function and homeostasis with architectural organization of the intestine.

Single-cell transcription site activation predicts chemotherapy response in human colorectal tumors.

Candidate gene and pathway approaches, and unbiased gene expression profiling, have identified marker signatures predictive of tumor phenotypes, such as drug sensitivity and invasive or metastatic potential. However, application of such information to evaluation of tumors in the clinic is limited by cell heterogeneity in the tumor. We have developed a novel method of fluorescence in situ hybridization (FISH) that can detect transcriptional activation of individual genes at their site in single cells in the interphase nucleus. A major obstacle in the treatment of colorectal cancer is relative insensitivity to the chemotherapeutic agent 5-Fluorouracil (5-FU). Here, we have developed a sensitive approach to predict relative sensitivity of colorectal cancer cells to 5-FU, using FISH with probes targeted to nascent mRNAs to measure the number of individual cells with active transcription sites for a panel of candidate genes. These results reveal that the transcriptional status of four key genes, thymidylate synthase (TYMS), MORF-related gene X (MRGX), Bcl2-antagonist/killer (BAK), and ATPase, Cu(2+) transporting beta polypeptide (ATP7B), can accurately predict response to 5-FU. As proof of principle, we show that this transcriptional profile is predictive of response to 5-FU in a small number of patient colon tumor tissues. This approach provides a novel ability to identify and characterize unique minor cell populations in the tumor that may exhibit relative resistance to chemotherapy.