Spectral biomarkers for chemoprevention of colonic neoplasia: a placebo-controlled double-blinded trial with aspirin.

OBJECTIVE: A major impediment to translating chemoprevention to clinical practice has been lack of intermediate biomarkers. We previously reported that rectal interrogation with low-coherence enhanced backscattering spectroscopy (LEBS) detected microarchitectural manifestations of field carcinogenesis. We now wanted to ascertain if reversion of two LEBS markers spectral slope (SPEC) and fractal dimension (FRAC) could serve as a marker for chemopreventive efficacy. DESIGN: We conducted a multicentre, prospective, randomised, double-blind placebo-controlled, clinical trial in subjects with a history of colonic neoplasia who manifested altered SPEC/FRAC in histologically normal colonic mucosa. Subjects (n=79) were randomised to 325 mg aspirin or placebo. The primary endpoint changed in FRAC and SPEC spectral markers after 3 months. Mucosal levels of prostaglandin E2 (PGE2) and UDP-glucuronosyltransferase (UGT)1A6 genotypes were planned secondary endpoints. RESULTS: At 3 months, the aspirin group manifested alterations in SPEC (48.9%, p=0.055) and FRAC (55.4%, p=0.200) with the direction towards non-neoplastic status. As a measure of aspirin's pharmacological efficacy, we assessed changes in rectal PGE2 levels and noted that it correlated with SPEC and FRAC alterations (R=-0.55, p=0.01 and R=0.57, p=0.009, respectively) whereas there was no significant correlation in placebo specimens. While UGT1A6 subgroup analysis did not achieve statistical significance, the changes in SPEC and FRAC to a less neoplastic direction occurred only in the variant consonant with epidemiological evidence of chemoprevention. CONCLUSIONS: We provide the first proof of concept, albeit somewhat underpowered, that spectral markers reversion mirrors antineoplastic efficacy providing a potential modality for titration of agent type/dose to optimise chemopreventive strategies in clinical practice. TRIAL NUMBER: NCT00468910.

Colonic Epithelial Angiotensin-Converting Enzyme 2 (ACE2) Expression in Blacks and Whites: Potential Implications for Pathogenesis Covid-19 Racial Disparities.

BACKGROUND: Covid-19 toll is disproportionate in Blacks although the mechanisms remain incompletely understood. From a biological perspective, several host proteins have received most attention as logical susceptibility targets. Specifically, angiotensin-converting enzyme 2 (ACE2) serves as the epithelial cell receptor and acts in concert with transmembrane protease serine 2 (TMPRSS2). Intriguingly, ACE2 can also suppress the inflammatory response and therefore may impact the severity of Covid-19 infections (from the exuberant immune response a.k.a. "cytokine storm"). We, therefore, assessed expression of ACE2 and TMPRSS2 in Blacks versus Whites. METHODS: Archived mucosal biopsies from colonoscopic biopsies of visually normal rectal mucosa without concurrent neoplasia or inflammation were used for this study. Total mRNA was isolated and subjected to real-time polymerase chain reaction for ACE2, and TMPRSS2 was assessed from non-Hispanic Blacks (n = 45) and non-Hispanic Whites (n = 38). GAPDH and beta-actin were used for normalization. Multivariable analysis was performed using Analyse-IT software. RESULTS: ACE2 and TMPRSS2 levels were not altered by gender, BMI, or age. ACE2 levels were lower in Blacks than Whites achieving statistical significance in multivariable (0.51-fold, p = 0.03) but not quite in univariable (p = 0.07) analysis. This downregulation was mirrored in TMRPSS2 in both univariable (p = 0.03) and multivariable analyses (0.41-fold, p = 0.02). Moreover, there was a strong correlation between ACE2 and TMPRSS2 levels (r-squared = 0.78). CONCLUSIONS: To our knowledge, this is the first report on racial differences inACE2 and TMPRSS2 mucosal expression. This may provide potential biological underpinnings for the disproportionately higher mortality of Covid-19 in Blacks and should spur future studies.

Single Nucleotide Polymorphism Facilitated Down-Regulation of the Cohesin Stromal Antigen-1: Implications for Colorectal Cancer Racial Disparities.

The biological underpinnings for racial disparities in colorectal cancer (CRC) incidence remain to be elucidated. We have previously reported that the cohesin SA-1 down-regulation is an early event in colon carcinogenesis which is dramatically accentuated in African-Americans. In order to investigate the mechanism, we evaluated single nucleotide polymorphisms (SNPs) for association with SA-1-related outcomes followed by gene editing of candidate SNP. We observed that rs34149860 SNP was significantly associated with a lower colonic mucosal SA-1 expression and evaluation of public databases showed striking racial discordance. Given that the predicted SNP would alter miR-29b binding site, we used CRISPR knock-in in CRC cells and demonstrated that the SNP but not wild-type had profound alterations in SA-1 expression with miR-29b inhibitor. This is the first demonstration of high-order chromatin regulators as a modulator of racial differences, risk alteration with SNPs and finally specific modulation by microRNAs.

Prevention of colonic neoplasia with polyethylene glycol: A short term randomized placebo-controlled double-blinded trial.

Chemoprevention represents an attractive modality against colorectal cancer (CRC) although widespread clinical implementation of promising agents (e.g. aspirin/NSAIDS) have been stymied by both suboptimal efficacy and concerns over toxicity. This highlights the need for better agents. Several groups, including our own, have reported that the over-the-counter laxative polyethylene glycol (PEG) has remarkable efficacy in rodent models of colon carcinogenesis. In this study, we undertook the first randomized human trial to address the role of PEG in prevention of human colonic neoplasia. This was a double-blind, placebo-controlled, three-arm trial where eligible subjects were randomized to 8g PEG-3350 (n = 27) or 17g PEG-3350 (n = 24), or placebo (n = 24; maltodextrin) orally for a duration of six months. Our initial primary endpoint was rectal aberrant crypt foci (ACF) but this was changed during protocol period to rectal mucosal epidermal growth factor receptor (EGFR). Of the 87 patients randomized, 48 completed study primary endpoints and rectal EGFR unchanged PEG treatment. Rectal ACF had a trend suggesting potentially reduction with PEG treatment (pre-post change 1.7 in placebo versus -0.3 in PEG 8+ 17g doses, p = 0.108). Other endpoints (proliferation, apoptosis, expression of SNAIL and E-cadherin), previously noted to be modulated in rodent models, appeared unchanged with PEG treatment in this clinical trial. We conclude that PEG was generally well tolerated with the trial failing to meet primary efficacy endpoints. However, rectal ACFs demonstrated a trend (albeit statistically insignificant) for suppression with PEG. Moreover, all molecular assays including EGFR were unaltered with PEG underscoring issues with lack of translatability of biomarkers from preclinical to clinical trials. This data may provide the impetus for future clinical trials on PEG using more robust biomarkers of chemoprevention. TRIAL REGISTRATION: ClinicalTrials.gov NCT00828984.

Topical polyethylene glycol as a novel chemopreventive agent for oral cancer via targeting of epidermal growth factor response.

Head and neck squamous cell carcinoma (HNSCC) is a major cause of morbidity and mortality underscoring the need for safe and effective chemopreventive strategies. Targeting epidermal growth factor receptor (EGFR) is attractive in that it is an early critical event in HNSCC pathogenesis. However, current agents lack efficacy or have unacceptable toxicity. Several groups have demonstrated that the over-the-counter medication, polyethylene glycol (PEG) has remarkable chemopreventive efficacy against colon carcinogenesis. Importantly, we reported that this effect is mediated through EGFR internalization/degradation. In the current study, we investigated the chemopreventive efficacy of this agent against HNSCC, using both the well validated animal model 4-NQO (4-nitroquinoline 1-oxide) rat model and cell culture with the human HNSCC cell line SCC-25. We demonstrated that daily topical application of 10% PEG-8000 in the oral cavity (tongue and cavity wall) post 4NQO initiation resulted in a significant reduction in tumor burden (both, tumor size and tumors/tumor bearing rat) without any evidence of toxicity. Immunohistochemical studies depicted decreased proliferation (number of Ki67-positive cells) and reduced expression of EGFR and its downstream effectors cyclin D1 in the tongue mucosa of 4NQO-rats treated with PEG. We showed that EGFR was also markedly downregulated in SCC-25 cells by PEG-8000 with a concomitant induction of G1-S phase cell-cycle arrest, which was potentially mediated through upregulated p21(cip1/waf1). In conclusion, we demonstrate, for the first time, that PEG has promising efficacy and safety as a chemopreventive efficacy against oral carcinogenesis.

Association of stem-like cells in gender-specific chemoprevention against intestinal neoplasia in MIN mouse.

This study was undertaken to examine the gender-sensitivity and chemopreventive responsiveness of celecoxib on intestinal stem-like cells as a biomarker of colon carcino-genesis, using the MIN mouse model. Male and female MIN mice (6-7-weeks old) were randomized to either control diet or to a diet supplemented with celecoxib (1,500 ppm). The animals were euthanized ten weeks later and the intestines were flushed and opened longitudinally to assess tumor count. Small intestinal segments were formalin-fixed and tissue sections were subjected to immunohistochemical evaluation of DCAMKL1, a known marker of stem-like cells. We found that in animals receiving control (AIN 76A diet) alone, female MIN mice had a higher polyp count than males (52.32 ± 13.89 vs. 35.43 ± 16.05; p<0.0005). However, compared to control diet groups, celecoxib supplementation caused a larger reduction in the number of polyps in females than their male cohorts (6.38 ± 1.43 vs. 12.83 ± 6.74; a reduction of 88% in females to 64% in males). Significant differences (p=0.013) were observed in the number of DCAMKL1-stained cells in the crypts of the wild-type (WT) (10.01 ± 1.07 stem cells per high powered field; HPF) compared to the MIN mice (24.15 ± 8.08 stem cells per HPF), illustrating increased stem-like cells in animals that are more prone to neoplasia. DCAMKL1 labeled stem-like cells were equal in number in the male and female groups receiving the control AIN 76A diet alone (females, 25.73 stem-like cells/HPF); males, 24.15 stem-like cells/HPF). However, females showed a greater reduction in the number of DCAMKL1-labeled stem-like cells with celecoxib supplementation than the respective males (16.63 ± 4.23 vs. 21.56 ± 9.06; a reduction of 35.4% in females to 10.7% in males). We conclude that a higher number of stem-like cells in the uninvolved mucosa paralleled tumorigenesis and mirrored greater chemopreventive responsiveness of female MIN mice compared to males.

Neo-angiogenesis and the premalignant micro-circulatory augmentation of early colon carcinogenesis.

Spectroscopic techniques have demonstrated that in the microscopically normal mucosa, there is an increase in mucosal micro-circulation in patients harboring neoplasia elsewhere in the colon (i.e. marker of field carcinogenesis). However, the physiological and molecular basis of this early increase in blood supply (EIBS) has not been elucidated. We, therefore, investigated the microvessel density (MVD) and angiogenic gene expression in the premalignant colonic mucosa from the well-validated azoxymethane (AOM)-treated rat experimental model of colon carcinogenesis. Fisher 344 rats were treated with AOM (15 mg/kg i.p.) or saline and euthanized 14 weeks later (a time-point that precedes carcinoma development). Colon sections were studied for MVD via immunohistochemical assessment for CD31 and location was compared with optical assessment of mucosal hemoglobin with low-coherence enhanced backscattering spectroscopy (LEBS). Finally, we performed a pilot real-time PCR angiogenesis microarray (84 genes) from the microscopically normal colonic mucosa of AOM and age-matched saline treated rats. AOM treatment increased MVD in both the mucosa and submucosa of the rats (125% increase in mucosa; p<0.007, and 96% increase in submucosa; p<0.02) but the increase was most pronounced at the cryptal base consistent with the LEBS data showing maximal hemoglobin augmentation at 200-225 µm depth. Microarray analysis showed striking dysregulation of angiogenic and anti-angiogenic factors. We demonstrate, for the first time, that neo-angiogenesis occurs in the microscopically normal colonic mucosa and was accentuated at the bottom of the crypt. This finding has potential implications as a biomarker for risk-stratification and target for chemoprevention.

Depth-resolved measurement of mucosal microvascular blood content using low-coherence enhanced backscattering spectroscopy.

Low-coherence enhanced backscattering (LEBS) spectroscopy is a light scattering technique which uses partial spatial coherence broadband illumination to interrogate the optical properties at sub-diffusion length scales. In this work, we present a post-processing technique which isolates the hemoglobin concentration at different depths within a sample using a single spectroscopic LEBS measurement with a fixed spatial coherence of illumination. We verify the method with scattering (spectralon reflectance standard and polystyrene microspheres) and absorbing (hemoglobin) phantoms. We then demonstrate the relevance of this method for quantifying hemoglobin content as a function of depth within biological tissue using the azoxymethane treated animal model of colorectal cancer.