For each cell, PWS microscopy generates a two-dimensional image of Ld(x,y) (Ld as a function of location within the cells). show representative microscope images of stained rectal colonocytes obtained from a control and a cancer patient, respectively. These images appear microscopically indistinguishable, suggesting no obvious alterations at microscopic length scales (>300 nm). However, when the pseudocolor maps of spatial distribution of rectal Ld are plotted, there appear regions with higher Ld (represented by red-color) indicative of nanoscale perturbations  for the colonocytes obtained from the cancer patient compared to control, indicating the nanoscale sensitivity of PWS. Furthermore, the augmentation of rectal Ld seems to be throughout the cell.
Representative rectal colonocytes from control and cancer patients
We first investigated whether the Ld
was sensitive to field-effect in histologically normal appearing rectal colonocytes obtained from various patient sub-types. In this study, there were N = 146 patients including controls (n = 72), patients harboring diminutive adenoma (polyp size < 5 mm, n = 14), non-diminutive-non-advanced adenoma (5 – 9 mm polyps, n = 20), advanced adenoma (polyp size ≥ 10 mm, high-grade dysplasia or >25% villous features, n = 15); patients harboring germline mutations for Lynch syndrome (n = 12) but without concurrent neoplasia and those having adenocarcinoma in their colon (n = 13). and show the overall results of rectal Ld
obtained from the reported patient population. As indicated in , there appears to be a progressive increase in Ld
that correlates with the risk of developing CRC: no neoplasia patients < non-advanced adenomas (most of which spontaneously regress) < advanced adenomas (a more aggressive precancerous lesion; CRC progression risk of 2-5% per year (24
)) < HNPCC patients (lifetime risk of CRC of 60-80% (25
)) < patients with frank CRC. Furthermore, demonstrates that there is no significant difference in the ΔLd
(Effect-size = 0.08, %Difference = 12.44%, P-value
~ 0.68) between control patients and those with diminutive adenoma. This result is consistent with the reported low risk of transformation (<0.1%) of diminutive adenoma to carcinoma(26
). However, there was a statistically significant ΔLd
(Effect-size = 0.64, %Difference = 42.44%, P-value
~ 0.0001) between control patients and those harboring intermediate size adenomas (5 – 9 mm polyps). ΔLd
increased further (Effect-size = 1.02, %Difference = 113.24%, P-value
~ 0.000006) for patients with advanced adenomas. These results indicate that Ld
is both significantly different and progressively increasing with higher polyp-size and hence with higher risk of developing CRC (26
). Importantly, they imply that PWS is sensitive to the colon field carcinogenesis (14
) and can quantify alterations in histologically “normal” appearing rectal colonocytes irrespective of the actual polyp location.
Results obtained from N = 146 patients using PWS microscopy
Table 1 highlights the statistical performance of the PWS analysis for all these category of patients compared to the controls. The %mean-differences, effect-size and the P-values are calculated on the log normalized Ld in order to obtain a normal distribution. (more ...)
We further assessed the PWS performance in patients with inherited genetic mutations which lead to familial colorectal cancer (CRC), as it accounts for approximately 15-20% of all CRCs (28
). Specifically, we investigated hereditary non-polyposis colorectal cancer (HNPCC, i.e., Lynch syndrome) cases which account for approximately 2-3% of all CRC patients (28
). This disease is mainly caused by germline mutations in the DNA mismatch repair genes such as MLH1, MSH2, MSH6, etc.(28
). PWS results from normal-appearing colonocytes demonstrated a two-fold increase and highly statistically significant ΔLd
(Effect-size = 1.17, % Difference = 184.5%, P-value
~ 0.000015) in these patients as depicted in . This sharp increase in Ld
parallels the reported elevated life-time risk (~ 70%) of developing CRC in these patients (28
). Moreover, ΔLd
is the highest (Effect-size = 1.42, % Difference = 281%, P-value
~ 0.0000006) between the control patients and cancer patients as shown in . This is expected as the cells obtained from cancer patients would have undergone the most nano-architectural alterations of all patient categories. Overall, and indicate that the disorder strength increase parallels the risk of developing CRC, from control patients to those with neoplastic lesions to patients with a proven history of colon cancer. This increasing trend suggests that rectal Ld
is correlated with the tumorigenicity of colon carcinogenesis and highlights the potential of PWS to risk-stratify patients for CRC based on “field-effect”, underscoring it as a promising pre-screening technique for colon cancer.
We next evaluated the diagnostic performance of PWS. In order to gauge the diagnostic capability of the PWS technique, we calculated the performance characteristics using the single parameter Ld
. It is important to note that we used a single marker to avoid any over-fitting of the dataset presented here. highlights the estimate of the AUROC using Ld
as the only diagnostic marker. The AUROC for PWS analysis of rectal brushings was 0.85 for advanced adenoma/HGD, and it improves further for higher risk patient populations. The AUROC was 0.89 for HNPCC and 0.92 for cancer patients. This result highlights the feasibility of the PWS technique for colon cancer screening and the satisfactory power of the single biomarker rectal Ld
. The values of the sensitivity and specificity depicted in are better (e.g., the sensitivity of FOBT, fecal-DNA is ~ 26% for HGD) than those of other existing screening techniques that are currently (albeit poorly) used as a pre-screen for colonoscopy (6
). In the future, these performance characteristics could be further improved by including other independent PWS-derived biomarkers.
shows the diagnostic performance of the single parameter, disorder strength (Ld) for various risk-groups.
One of the challenges with CRC screening is the early detection of proximal colonic neoplasia. There is a growing debate about the sensitivity of colonoscopy to proximal lesions (30
). Hence, we tested the sensitivity of PWS to proximal adenomas. provides information about the location of the polyp and its effect on Ld
. It appears from that there is a statistically non-significant difference in ΔLd
(Effect-size = 0.30, P-value
~ 0.15) between patients having proximal and distal lesions. There were 19 patients having proximal polyps of size > 5 mm and 16 patients with similar distal lesions. These results suggest that PWS is equally sensitive to both proximal and distal lesions. This is an important result with respect to sub-optimal efficacy of colonoscopy (30
) and flexible sigmoidoscopy (8
) to proximal lesions. We believe that PWS can be a handy tool for clinicians in successfully diagnosing proximal lesions and can potentially reduce the polyp miss rate. Additionally, pre-screening using PWS could filter patient populations that might benefit from further invasive colonoscopic investigation.
depicts that Ld from rectal colonocytes is equally sensitive to both, the patients having proximal lesions (n = 19) and distal lesions (n =16) of size > 5mm with a non-significant P-value ~ 0.15.
For the control group (n = 72) in our study, we included patients harboring hyperplastic polyps (n = 7) and patients having diverticulitis (n = 8) in their colon. We evaluated the confounding effect of these subtypes of patients on the PWS performance. However, there seems to be no statistically significant difference (Effect-size = 0.18, P-value ~ 0.25) between Ld values of the patients with non-neoplastic lesions and those with clean colons. Hence, we combined all these patients in the ‘control’ group.
We next studied the role of confounding demographic risk factors such as age, gender, smoking and drinking history on the sensitivity of the measured biomarker. Age has been implicated as one of the key risk factors for colonic neoplasia and there have been a variety of age-related changes in colonic mucosa (such as methylation (31
)). We therefore performed ANCOVA analysis and noted no significant confounding with age (P
= 0.54 for Ld
). As outlined in , smoking and drinking history also did not have any confounding effect on Ld
= 0.57 for current smokers and P
= 0.99 for drinking). Similarly, male gender is a well-established risk factor for colonic neoplasia(32
). However, ANCOVA analysis indicated that there was no significant confounding with gender (P
= 0.29 for Ld
). Overall, the non-significant ANCOVA P-values suggest that Ld
is not confounded by age, gender, smoking or drinking patterns.
shows the impact of demographic factors on the single biomarker
The next question we addressed was the performance of Ld in a prospective study. Although we understand that we have a modest dataset to make any definitive conclusions, we still wanted to gauge the diagnostic power of our approach when tested on an independent training and testing set for any patient having an adenomatous polyp of size ≥ 10 mm or with tubulovillous features. We specifically selected advanced adenoma patients as they comprise the most clinically relevant screening population. The training set was comprised of a subset of n = 87 patients (wherein 72-controls and 15-advanced adenoma) out of the N =146 total patients. However, the validation set (N = 39 patients with 14-control and 25-advanced adenoma) was an independent enriched dataset for case-control recruited from a second clinical site We developed a cutoff based on the Ld values of the training set and applied the same to the validation set. The estimates of sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) based on this threshold are listed in . As summarized in , we obtained promising performance characteristics for the training set (sensitivity = 73%, specificity = 78%, PPV = 41%, NPV = 93%). The 95% confidence intervals for all these parameters were: Sensitivity = 0.733 [0.48 to 0.891]; Specificity = 0.778 [0.669 to 0.858]; PPV = 0.407 [0.245 to 0.593]; NPV = 0.933 [0.841 to 0.974]. We then applied the same Ld threshold to the validation set which yielded sensitivity = 74% and specificity = 83%, similar to the training set and further supporting the robustness of our conclusions We again emphasize that this performance is based on a single biomarker (Ld) and it is probable that inclusion of additional PWS-measured markers would further improve the performance characteristics.
highlights the performance of Ld on an independent training (n = 87) and testing set (n = 39) developed for controls and patients having advanced adenoma