We provide the first demonstration that light scattering signatures in histologically normal rectal mucosa are altered in patients harboring neoplasia elsewhere in the colon. Individually, three of four parameters from the LEBS signal correlated with the presence of advanced neoplasia (ANOVA P
< 0.05). The weighted sum of these parameters, the LEBS marker, showed dramatic alterations in patients with biologically significant adenomas (>5 mm), and this relationship was not confounded by the presence of common CRC risk factors. These studies are consistent with both our reports in preclinical models that LEBS markers from predyplastic epithelium can predict future neoplasia (20
) and also with a pilot human study, which noted that a single spectral marker derived from an earlier generation technology (four-dimensional elastic light-scattering fingerprinting; ref. 24
) was decreased in the midtransverse colon of patients harboring adenomatous polyps (25
). The current report is a major step forward using a more accurate technology, markedly larger data set, four distinct spectral parameters, and most importantly, providing the first evidence of feasibility in a clinically relevant application (rectal mucosal assessment). Our future clinical vision is that rectal LEBS analysis could be performed with a fiberoptic probe inserted into the rectum (analogous to a rectal thermometer). If the LEBS marker threshold level is not met, the patient could forego immediate more intrusive CRC screening, whereas if the threshold were surpassed, immediate colonoscopy would be recommended.
We believe that the biological underpinning of the altered light scattering signatures in patients harboring adenomas is the microarchitectural consequenecs of the genetic/epigenetic perturbations that are the hallmarks of field carcinogenesis. Although the field effect concept is widely used in current clinical practice, the current markers are inadequate. For instance, flexible sigmoidoscopy is commonly used (1.2 million screening exams performed in 2002; ref. 26
), identification of the sentinel adenoma (the intermediate biomarker for proximal neoplasia) is inadequate especially in women (identifying ~1/3; ref. 27
). Earlier focal neoplastic lesions (e.g., rectal aberrant crypt foci) may also be somewhat insensitive to proximal neoplasia (13
). Therefore, research interest has focused on detecting diffuse field carcinogenesis in the predysplastic (histologically normal) mucosa. The techniques used have ranged from cellular markers (apoptosis and proliferation; ref. 15
) to more powerful technologies such as microarray (16
) or proteomic analysis (17
). These approaches, while providing corroboration of the approach of detecting a field effect in the histologically normal distal colonic mucosa, are not suitable for population screening because of issues related to accuracy and practicality. The power of LEBS for identifying field carcinogenesis is not only its sensitivity for important lesions but also that the “bench-to-bedside” transition seems to be feasible.
These observations have been made possible by a breakthrough in optics technology from our laboratory (18
). Our group first described EBS in tissue by using a low-coherence light to overcome challenges of speckle, narrow peak width, and lack of spectroscopic information. The premise of using elastically scattered light for tissue characterization is based on the fact that light scattering depends on the spatial variations of tissue refractive index, which is proportional to the local concentration of tissue constituencies (18
). LEBS signatures are governed not simply by the size and shape of scattering particles but also the relationship with the internal cellular milieu. Indeed, our work with two well-validated models of colon carcinogenesis (azoxymethane-treated rat and the MIN mouse) indicated that LEBS markers from the histologically normal mucosa had remarkable accuracy in identifying risk for future neoplasia (20
). Thus, LEBS optical markers can be exquisitely sensitive to the earliest genetic/epigenetic changes of field carcinogenesis (20
Preliminary performance characteristic estimates for the rectal LEBS analysis were very encouraging for advanced adenomas. The cut-points were designed to maximize sensitivity to have an outstanding negative predictive value. Because we envision use of rectal LEBS as a precolonoscopic screening, the negative predictive value is of paramount importance. It is critical that all patients who are deemed to be negative by LEBS in actuality do not harbor significant neoplasms. We believe that a lower positive predictive value is acceptable because without the prescreening tests, all these subjects should have undergone colonoscopy. Furthermore, the LEBS results need to be kept in perspective of other tests that have been advocated to prescreen for colonoscopy. For instance, a guaiac-based fecal occult blood test, one of the most common colon cancer screening test, had a sensitivity of 10.8% for advanced neoplasia (cancer or advanced adenoma; ref. 3
). Fecal DNA analysis has a minimum improvement (18.2% sensitivity for advanced neoplasia) but at a large increase in costs (3
). Studies on novel markers of field carcinogenesis in the colon (genomic and proteomic) have generally been more phenomenological and not geared for performance characteristics. However, one may glean insight about the promise of his approach through comparable studies in other malignancies. For instance, using a similar approach in lung cancer (microarray analysis from the endoscopically normal right mainstem bronchus) yielded a sensitivity and specificity of 80% and 84%, respectively (29
). These performance characteristics for our field carcinogenesis-based approach would seem to be clinically relevant as judged by the fact that although computed tomography colography (virtual colonoscopy) had a sensitivity for advanced adenoma of 84% (per patient analysis of 90%) with a positive predictive value of only 23% (30
), this was sufficient to be sanctioned for population screening (2
). Others reports have suggested that computed tomography colography may be more useful as a precolonoscopic risk stratification, although the need for bowel purge, radiation exposure, and expense may make this approach less desirable (31
). When viewed in this context, rectal LEBS may be quite attractive for CRC population screening given its minimally intrusive nature and accuracy. Moreover, our preclinical data would suggest that this approach may also have utility in assessment of response to chemopreventive agents or determination of risk in individuals with a family history of CRC, although clinical confirmation is clearly required (32
Our results indicated that rectal LEBS was altered in patients harboring advanced adenomas. For those with intermediate adenomas (5–9 mm), the effect size seemed reasonable, which may be relevant given that these lesions have a 1 in 15 chance of harboring advanced features (33
). The clinical implications for the insensitivity for diminutive adenomas are unclear. One could argue that the risk of advanced features in these lesions are negligible (34
). On the other hand, the realization that lesions such as flat and depressed lesion can harbor advanced features at smaller diameter is of concern (35
). Although this remains to be resolved, it bears emphasis that computed tomography colography received approval for CRC screening, yet this test is, in general, unable to detect diminutive adenomas (2
Rectal LEBS measurements were sensitive to lesions in both the distal and proximal colon, although the magnitude of alterations was clearly greater in the distal colon. This may simply be a distance effect. On the other hand, this may reflect regional biological differences with the proximal tumors more likely to be microsatellite unstable (36
). Moreover, because there is emerging evidence that some of the biological basis of field changes (e.g., promotor hypermethylation in the uninvolved mucosa) may be distinct between proximal and distal neoplasia, it seems reasonable to conjecture that the rectal LEBS marker may reflect these differences (36
For rectal LEBS analysis to be a practical screening test, it would need to be performed in situ
and preferably without colonic preparation. Our group has recently reported that for other markers of field carcinogenesis (i.e., microvascular blood content), in situ
testing seemed to improve performance characteristics over the ex vivo
). Preliminary studies using a recently designed endoscopically compatible LEBS probe indicates that not only can LEBS markers be assessed from the rectum, but the ability to predict proximal neoplasia seems promising (39
). Larger scale studies using patients with and without colonic purges are currently ongoing.
There are several limitations of this study. The number of advanced adenomas in this screening population was modest. In any biomarker study, it is mandatory to use a robust independent validation set before pronouncements over performance characteristics can be confidently made. While awaiting completion of a comprehensive validation study, we were able to partially mitigate these concerns by analyzing a different cohort. As described, this small convenience set (n = 51) unfortunately lacked advanced adenomas. However, it was reassuring that the AUROC for total adenomas was almost comparable between the initial and validation data sets. In addition, we performed a leave-one-out cross validation for advanced adenomas in the initial data set, which further assuages these concerns.
Another issue is the modest specificity (80% for advanced adenomas) in this trial. An important caveat is that the gold standard (colonoscopy) is clearly not infallible. Tandem colonoscopy studies estimate that 22% of all adenomas are missed (4
). These can occur for advanced adenomas as suggested by a ~6-fold difference in detection rate among well-trained endoscopists (40
). Therefore, our preliminary report on performance characteristics is probably an underestimate of the true accuracy of rectal LEBS for both total and advanced adenomas. Additionally, endoscopic adenoma size estimation may be somewhat inaccurate although this should not bias our results (41
). The issue of whether rectal LEBS reflects immediate or long-term risk is unclear. There did seem to be a slight association with tobacco and alcohol use, which are factors that our group (23
), and others (43
) have reported to be modest risk factors for CRC. Our studies on the effect of previous adenomas are inconclusive but potentially suggest contributions from both longer term field carcinogenesis and tumor-related factors analogous to our data for microvascular blood content (38
). Other groups have shown that rectal apoptosis rate predicts future development of neoplasia, suggesting that the markers of field carcinogenesis may reflect immediate as well as long-term risk (44
). Thus, the utility of rectal LEBS for postpolypectomy surveillance will need to be determined.
Finally, it bears mentioning that the biological determinants of these alterations in spectral markers remain incompletely understood. One could argue that the molecular pathogenesis of many biomarkers (serum proteomics, microarrays, etc.) is poorly understood, but that this is irrelevant to its cancer screening applications. In principle, the only requirement is an excellent correlation between biomarker level and presence of the disease of interest (45
). On the other hand, this approach, although clinically appropriate, seems somewhat intellectually unsatisfying. We, therefore, have conducted preliminary studies demonstrating that specific genetic alterations in colon carcinogenesis can alter optical nanoscale signatures both in cell culture (46
) and the MIN mouse intestinal mucosa (47
). Systematic studies are under way to more precisely elucidate the molecular and ultrastructural etiologies of the LEBS spectral marker alterations in field carcinogenesis.
In conclusion, we show, for the first time, that microarchitecture of the uninvolved rectal mucosa was altered in subjects harboring neoplasia elsewhere in their colon. These alterations were detectable by a novel, highly sensitive optical technique, LEBS. This represents a proof-of-principle that optical interrogation of the endoscopically normal rectal mucosa may allow risk-stratification for colon neoplasia. In the future, one could potentially envision that during the annual rectal examination, the primary care physician could insert this thin, flexible probe to determine the need/timing for more intrusive tests such as colonoscopy. Furthermore, this work may herald the role for biomedical optics as a powerful cancer risk-stratification tool.