Evaluation of Quantitative Digital Pathology in the Assessment of Barrett Esophagus-Associated Dysplasia.

AJCP / Original Article

Evaluation of Quantitative Digital Pathology in the Assessment of Barrett Esophagus–Associated Dysplasia Soufiane El Hallani,1 Martial Guillaud,2 Jakoda Korbelik,2 and Esmeralda Celia Marginean, MD, FRCPC, FCAP1 From the 1Department of Pathology and Laboratory Medicine, University of Ottawa, Ottawa, ON, Canada, and 2Integrative Oncology—Cancer Imaging Unit, British Columbia Cancer Research Centre, Vancouver, BC, Canada.

CME/SAM

Key Words: Barrett esophagus; Dysplasia; Quantitative pathology Am J Clin Pathol July 2015;144:151-164 DOI: 10.1309/AJCPK0Y1MMFSJDKU

ABSTRACT Objectives: Barrett esophagus (BE) is a precursor lesion that confers an increased risk of esophageal adenocarcinoma. Two issues confront the diagnosis of patients with BE: (1) sampling error at the time of endoscopy and (2) variability among pathologists in grading dysplasia. The purpose of our study was to evaluate quantitative digital pathology (QDP) as a marker of dysplasia and stratification from low-grade to high-grade dysplasia to intramucosal adenocarcinoma in BE. Methods: Sixty-one esophageal biopsy specimens with BE were selected and divided into six groups according to the dysplasia grade. QDP image analysis was carried out by an in-house automated quantitative system on sections. The values of 110 nuclear features that analyze the morphology and chromatin texture were generated for each nucleus. Results: A progressive correlation was found between nuclear morphometric features and chromatin features with BE dysplasia. The chromatin texture was the best discriminator of the class diagnosis. There was a significant difference between the chromatin features of isolated low-grade dysplasia vs low-grade dysplasia that was associated with higher grade lesions in other biopsy tissue fragments. Conclusions: QDP is a promising tool in the new era of digital pathology. Pending clinical validation studies, analysis of chromatin texture could contribute to the differential diagnosis of BE class and the detection of concomitant high-grade lesions if not sampled.

© American Society for Clinical Pathology

Upon completion of this activity you will be able to: • describe the appropriate procedure for Barrett esophagus (BE) surveillance and management. • list the major limitations of endoscopic biopsies in the diagnosis and grading of BE dysplasia. • correlate the objective measurement of nuclear characteristics by quantitative digital pathology with the different class diagnosis of BE. • discuss the role of quantitative digital pathology in predicting the concomitant presence of high-grade dysplasia or carcinoma when a diagnosis of low-grade dysplasia is made based on routine H&E histopathology. The ASCP is accredited by the Accreditation Council for Continuing Medical Education to provide continuing medical education for physicians. The ASCP designates this journal-based CME activity for a maximum of 1 AMA PRA Category 1 Credit ™ per article. Physicians should claim only the credit commensurate with the extent of their participation in the activity. This activity qualifies as an American Board of Pathology Maintenance of Certification Part II Self-Assessment Module. The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. Exam is located at www.ascp.org/ajcpcme.

In the United States and other Western countries, there has been a remarkable change in the epidemiology of esophageal cancer over the past 50 years. Adenocarcinoma of the esophagus and gastroesophageal junction has replaced squamous cell as the most common type of esophageal cancer in the United States, and the incidence of esophageal adenocarcinoma is increasing faster than that of any other malignancy.1 Barrett esophagus (BE) is defined by metaplastic glandular changes to the distal esophagus, including goblet cells (intestinal metaplasia). Controversy exists whether the definition should be limited to intestinal-type glands with goblet cells or expanded to include non–goblet cell columnar epithelium (cardia- and fundic-type mucosa). Only intestinal metaplastic changes have been clearly linked to an increased risk of malignant progression, with a reported annual risk

Am J Clin Pathol 2015;144:151-164 151 DOI: 10.1309/AJCPK0Y1MMFSJDKU

El Hallani et al / QDP in Assessing BE-Associated Dysplasia

A

C

of esophageal adenocarcinoma of about 0.5% per year in patients with intestinal metaplasia of the esophagus.2-4 For this reason, both the American Gastroenterological Association and the American College of Gastroenterology currently recommend that although columnar-type mucosa can be recognized during endoscopy, the presence of intestinal metaplasia must be confirmed by biopsy before rendering a diagnosis of BE. BE is a precursor lesion that confers a 30- to 100-fold increased risk of esophageal adenocarcinoma above that for the general population, with incidence rates of 0.4% to 2% per annum in nondysplastic BE.2,5,6 Progression appears to occur through a metaplasia-dysplasia-carcinoma sequence.5,7-9 Worldwide, there are two classification systems used for BE dysplasia. One was proposed in 1983 by the Inflammatory Bowel Disease/Dysplasia Morphology Study Group, which classified dysplasia as negative, indefinite, or positive (lowor high-grade dysplasia), intramucosal adenocarcinoma 152 Am J Clin Pathol 2015;144:151-164 DOI: 10.1309/AJCPK0Y1MMFSJDKU

B

❚Image 1❚ Barrett esophagus, low end of dysplastic spectrum. The photographs show the region of interest that served to make the diagnosis and quantify the nuclear features by image analysis. A and B, Barrett esophagus, negative for dysplasia. There is columnar cell metaplasia, including mucin-filled, blue-tinted goblet cells. The glands are well spaced with abundant intervening lamina propria, and the nuclei are regular, smooth, and basally located (H&E, ×20). C, Indefinite for dysplasia. Nuclear stratification and hyperchromasia of surface and glandular epithelium are noted in a background of chronic active inflammation; rare neutrophils are seen infiltrating surface epithelium. Key features of regeneration in this case include low nuclear/ cytoplasmic ratio, small nucleoli, and relative preservation of cell polarity (H&E, ×20).

(IMC), and invasive adenocarcinoma (INV), which is the system used most commonly in North America ❚Image 1❚ and ❚Image 2❚.10 Recently, the World Health Organization proposed that the term dysplasia be replaced by intraepithelial neoplasia, but this new term has yet to gain popularity. The second classification system is the Vienna classification system,11 similar to the one proposed by the Inflammatory Bowel Disease/Dysplasia Morphology Study Group, except that it uses the term noninvasive neoplasia, instead of lowgrade dysplasia (LGD) or high-grade dysplasia (HGD), and also uses the term suspicious for invasive carcinoma for lesions that show equivocal cytologic or architectural features of tissue invasion.12,13 Surgical treatment is indicated only for HGD and carcinoma.14 Patients diagnosed with BE are recommended to be enrolled in endoscopic surveillance programs with four quadrant biopsies for every 2 cm of the Barrett mucosa.15 Presently, the diagnosis of BE is done by routine light microscopy on H&E-stained formalin-fixed



D

E

F

❚Image 1❚ (cont) D, Indefinite for dysplasia. An area of ulceration is present, devoid of surface epithelium; therefore, surface maturation cannot be evaluated. In addition, adjacent epithelium shows mild nuclear atypia (H&E, ×10). E, Low-grade dysplasia. Overall, there is mild glandular crowding. The cells show mucin depletion, with rare goblet cells still present. The nuclei of glandular and surface epithelium are pencil-shaped, hyperchromatic, and stratified but mostly limited to the basal half of the cell cytoplasm (H&E, ×10). F, Low-grade dysplasia. Other areas show larger nuclei and mitotic figures but still preserved nuclear polarity (H&E, ×20).

tissue. Current guidelines recommend that the diagnosis of HGD and higher should be confirmed by an expert gastrointestinal (GI) pathologist.15 Two significant issues confronting the management of patients with BE include sampling error at the time of endoscopy, since the biopsies sample only a small fraction of the mucosa, and poor agreement among pathologists grading BE dysplasia. Significant intra- and interobserver variability in the interpretation of biopsy specimens has been well documented, even between expert GI pathologists, especially at the lower end of the BE dysplasia spectrum (eg, negative vs indefinite for dysplasia vs LGD16,17). There is much interest in developing molecular biomarkers in BE, both to predict which patients may develop carcinoma (and therefore be offered surgical therapy with curative intent) and to aid prognostication and guiding surveillance intervals following therapy. Abnormalities in DNA ploidy have been studied in BE; they are a consequence of genomic instability that has been


shown to predict future cancer risk in nondysplastic BE.18 DNA ploidy can be measured by image cytometry or by flow cytometry.19 Image cytometry uses digital images of Feulgen-stained nuclei and is an accurate method to estimate DNA content, comparable with flow cytometry.20 At the tissue level, similar technologies such as quantitative digital pathology (QDP)21 can provide precise measurement of different nuclear characteristics, with each being recognized as a hallmark of dysplastic and neoplastic changes. The QDP algorithm breaks down the components of the nuclei into multiple quantifiable and numeric units at the pixel level, allowing the investigator to objectively standardize morphometric criteria for accurate diagnosis and examine associations of such quantifiable changes with the risk of malignant progression.21 Using different image analysis and statistical methods, the potential of image analysis to measure the grade of dysplastic lesions has been demonstrated in different tissues types, such as skin, ovary, prostate, or



A

B

❚Image 2❚ Barrett esophagus, high end of dysplastic spectrum. A, High-grade dysplasia. On low power, marked architectural atypia is noted, with crowded glands, back-to-back, with little intervening stroma (H&E, ×4). B, On higher power, marked nuclear atypia is noted involving glandular and surface epithelium, with mucin depletion; hyperchromatic, large, round nuclei; high nuclear/cytoplasmic ratio; and numerous mitoses toward the luminal surface (H&E, ×10).

breast.22-25 QDP is especially useful in so-called continuous lesions, in which interobserver and intraobserver disagreement is considerable. Interestingly enough, a reference study has demonstrated that computerized morphometry is a valid tool for determining the degree of BE-associated dysplasia and predicting the progression of HGD into INV.23 In the evolving field of image analysis pathology, additional data are much needed prior to any potential clinical consideration. The goal of this study was to evaluate the contribution of a different and independent in-house QDP system in (1) measuring the nuclear changes that occur with the progression of BE, from nondysplastic to LGD to HGD to IMC to INV (nuclear morphometric and chromatin texture); (2) differentiating between nondysplastic BE, BE indefinite for dysplasia, and BE with LGD, categories that are subject to significant interobserver variation; and (3) discriminating between isolated LGD and LGD that is associated with and/ or has the potential to progress to higher grade lesions.

Materials and Methods Case Selection A total of 61 esophageal biopsy specimens with BE were retrospectively selected from the Department of Pathology and Laboratory Medicine archives of The Ottawa Hospital. BE was defined by intestinal metaplasia with goblet cells present in the distal esophagus. Goblet cells are recognized by a large cytoplasmic vacuole filled with blue-tinted mucin.

154 Am J Clin Pathol 2015;144:151-164 DOI: 10.1309/AJCPK0Y1MMFSJDKU

All cases were signed out by the study pathologist with GI expertise (E.C.M.). The cases showing HGD and IMC were confirmed at the time of sign-out by a second specialized GI pathologist, according to current guidelines.15 All diagnoses were verified again at the time of selection by the study pathologist (intraobserver agreement). In case there was a discrepancy between the original diagnoses and the latest diagnosis, the latest was used, based on the current diagnostic criteria.10,17,26 Six cases originally diagnosed as indefinite for dysplasia (IND) were recategorized as negative for dysplasia, and two cases originally diagnosed as LGD were downgraded to IND. The degree of dysplasia was determined by evaluating the cytology (nuclear and cytoplasmic features), architecture (relationship of glands and lamina propria), degree of surface maturation (comparison of nuclear size within crypts to nuclear size at the mucosal surface), and interpreting these findings in conjunction with the amount of background inflammation. Features of each category of dysplasia are described below, according to the currently used diagnostic guidelines.12,16,17 Negative for Dysplasia Biopsy specimens that are negative for dysplasia can have a minimal amount of cytologic atypia but retain normal architecture, with round glands, similar in size and shape, with abundant lamina propria between glands. Surface maturation is present, defined by a low nuclear/cytoplasmic ratio at the mucosal surface and regular nuclei, basally situated in a single row, with smooth membranes. If mitoses are present, they are within the basal compartment (Images 1A and 1B).



C

D

E

F

❚Image 2❚ (cont) C and D, Intramucosal adenocarcinoma. Marked architectural atypia, with cribriform glands and intraluminal necrosis. Single markedly atypical cells are infiltrating the lamina propria; the glands or single cells do not reach the muscularis mucosae (H&E, ×20). E and F, Intramucosal adenocarcinoma. Markedly distorted, angulated glands are invading the lamina propria and infiltrating in between layers of the muscularis mucosae (level M3). Invasion beyond the muscularis mucosae into submucosa cannot be excluded in a mucosal biopsy specimen (H&E, ×20).

IND

The IND category is applied to biopsy specimens in which the changes seen cannot be definitively described as reactive or neoplastic. It is most often used in three situations: in the presence of pronounced active inflammation and/or ulceration; technical issues, with loss of surface epithelium, tangential sectioning, or cautery artifacts; and dysplasia-like changes present only in the bases of crypts, with surface maturation.12 Cytologic atypia is characterized by hyperchromasia, overlapping nuclei, and irregular nuclear borders, and nuclear stratification can be seen in the deep glands or the sides of villiform structures, while the surface epithelium is free of atypia. The architecture should


be largely normal with, at most, minimal gland crowding. Surface maturation is present (Images 1C and 1D). Although numerous GI pathologists still categorize dysplasia-like changes present only in the bases of crypts as IND, there is evidence that these type of lesions are associated with dysplasia or adenocarcinoma, and combined with evidence of proliferative and molecular abnormalities in affected patients, this suggests that these lesions may be a true subtype of dysplasia.27 LGD The most important feature of LGD is cytologic atypia extending to the mucosal surface and either minimal or



absent surface maturation. Severe architectural distortion is not a feature, although mild gland crowding with decreased intervening lamina propria can be seen. Mitoses may be increased, but no atypical forms should be seen. Inflammation is usually minimal. One important note: although cytologic atypia is a key finding, nuclear polarity is preserved (nuclei are perpendicular on the basement membrane) (Images 1E and 1F). HGD In HGD, the cytologic changes are severe, with markedly enlarged nuclei at the surface, pronounced pleomorphism, and at least focal loss of nuclear polarity. Surface maturation is lost. Mild to marked architectural distortion is a frequent finding, with crowded glands, with little intervening lamina propria, focal budding, and/or cribriform glands. There should be no evidence of invasion into the lamina propria. Mitoses are increased and atypical mitoses may be seen. Inflammation is minimal or absent (Images 2A and 2B). IMC IMC is defined as invasion of the lamina propria, either by single cells or by small clusters of tightly compact backto-back glands (Image 2C). A complex gland-in-gland or “cribriforming” pattern may be present, with or without luminal necrosis (Image 2D). The glands are lined by epithelium with marked nuclear atypia, similar to HGD. Desmoplasia is rarely present in IMC. The distinction between HGD and IMC may be difficult and is often clinically relevant, since lymphatic vessels are present in the lamina propria and there is a 5% to 8% risk of lymph node metastases.28 In well-oriented biopsy specimens or endoscopic mucosal resection specimens, the depth of invasion into the lamina propria may be assessed. According to the Japanese classification of esophageal cancer, HGD is equivalent to carcinoma in situ (or level M1), tumor infiltrating the lamina propria without reaching the muscularis mucosae (MM) represents T1a-LP (or level M2), and tumor infiltrating the MM represents T1a-MM (or level M3) (Images 2E and 2F).29 Our selected cases were divided into six groups16,17,30: (1) negative for dysplasia (n = 10); (2) IND due to associated active inflammation and/or loss of surface epithelium with adjacent epithelial atypia (n = 9); (3) LGD (n = 14); (4) HGD (n = 10); (5) IMC, defined as invasion into the lamina propria without clearly reaching the MM or MM not present in the biopsy specimen (n = 9); and (6) IMC invading into the MM (INV; n = 9). Among the LGD group, there were two comparative subgroups: (a) isolated LGD (n = 6) and (b) LGD with adjacent higher grade lesions, either within the same tissue fragment or in another tissue fragment of the same sample, as follows: HGD (n = 4), IMC without invasion of the MM (n = 3), and IMC invading the MM (n = 1).


The tissue was fixed in buffered formalin and embedded in paraffin blocks. Two 5-µm serial sections were cut from each tissue block; one section was stained with H&E for conventional interpretation, and the other was stained with a quantitative stoichiometric Feulgen-Thionin stain, in which the amount of the dye bound per nucleus was proportional to its DNA content.31 QDP Analysis Software All the Feulgen-Thionin–stained slides were analyzed using our previously described in-house high-resolution image analyzer system (Getafics; BCCA, Vancouver, Canada).21,32 This software was specifically designed for semiautomatic analysis of tissue sections. Feulgen-Thionin–stained nuclei were measured with a monochromatic light at a wavelength of 600 nm using a 20×0.75-NA Plan APO objective lens. With a printout of the diagnostic area on hand, a cytotechnologist located the same area in the Feulgen-Thionin–stained slide as the region of interest (ROI) of the H&E slide. As illustrated, the cytotechnologist delineated the basal membrane and the surface of the superficial epithelium ❚Image 3A❚, which defined the ROI. Image Analysis A threshold algorithm was applied to the image ❚Image 3B❚, followed by a segmentation algorithm to separate touching and overlapping. A manual correction of the nuclear segmentation was made for touching objects ❚Image 3C❚. Autofocusing and edge relocation algorithms were applied to the nuclei to place the edge of the object precisely and automatically along the contour of the highest local gray-level gradient. The digital gray-level images of these nuclei were stored in a gallery ❚Image 3D❚ and analyzed using computer calculations ❚Image 3E❚. Quality Control The cytotechnologist manually reviewed each object in an image gallery of all the selected nuclei and removed any object that did not fulfill the minimum requirements (bad nuclear mask, out-of-focus image, pale nucleus, pyknotic nucleus). Nuclear Feature Calculation Nuclear features were extracted from the digitized images of the Feulgen-Thionin–stained nuclei ❚Table 1❚. In total, 103 cytometric features were calculated.24,33 Morphologic features described the nuclear size, shape, and border irregularities. The five photometric features estimated the absolute intensity and optical density of the nucleus and the intensity distribution characteristics. DNA



A

B

D

C

E

❚Image 3❚ Getafics (BCCA, Vancouver, Canada) software user interface. A, Region of interest (ROI) is manually delineated according to pathologist diagnosis: red line represents the basement membrane, and pink line represents the epithelium surface. B, Nuclei are automatically segmented by thresholding (blue) and (C) selected within the ROI. D, After automatic collection of individually focused images, an interactive correction of segmentation errors and selection of only nonoverlapping nuclei are performed as quality control. In this example, graphic representation of one of 110 nuclear features assessed in this study: DNA index. E, Graphic representations of three of the 110 nuclear features assessed in this study, all of which measure DNA distribution in the nucleus. Left, OD-skewness measures whether the nucleus is dark with light areas or light with dark areas; middle, Fractal_area1 measures heterochromatin vs euchromatin organization (ie, large intensity contrast between highly condensed chromatin and noncondensed chromatin); right, Long90_Run measures the fraction of nuclear diameter one can travel before an intensity change is encountered.

amount was the raw measurement of the integrated optical density (IOD) from which all the photometric features were derived. The IOD norm was the mean value of the DNA amount of a reference population. The DNA index was the normalized measure of the IOD of the object (ie, the DNA amount divided by IOD norm). We used the average DNA amount for the epithelial cell population as the IOD norm value.


Texture Features Discrete texture features were based on thresholded segmentation of the object into regions of low, medium, and high optical density. Markovian texture features characterized gray-level correlation between adjacent pixels in the image. Non-Markovian texture features described the texture in terms of local maxima and minima of gray-level differences in the object. Fractal texture features described the



❚Table 1❚ Quantitative Nuclear Features Measured on Each Cell Category Morphologic features Size Shape Boundaries DNA content Texture features Discrete texture

Features mean_radius, variance_radius, maximum_radius eccentricity, sphericity, elongation, compactness, inertia_shape low_freq_fft, freq_low_fft, harm01-32_fft DNA_index, OD_max, OD_var, OD_skew, OD_kurt

low, medium, high DNA amount; low, medium, high DNA area; low, medium, high, medium_high DNA compactness; low, medium, high, medium_high DNA average distance; low, medium, high density object; low, medium, high center mass; low_vs_medium, low_vs_high, low_vs_medium-high DNA Markovian texture entropy, energy, contrast, correlation, homogeneity, cl_shade, cl_prommence Non-Markovian texture density_light_spots, density_dark_spots, center_of_gravity, range_extreme, range_average Fractal texture fractal_area1, fractal_area2, fractal_dimension Run-length texture short_runs_mean, short_run_stdv, short_run_min, sort_run_max, long_runs_mean, long_run_stdv, long_run_ min, long_run_max, gray_level_mean, gray_level, stdv, gray_level_min, gray_level_max, run_length_mean, run_length_stdv, run_length_min, run_length_max, run_percent_mean, run_percent_stdv, run_percent_min, run_percent_max

❚Table 2❚ Photographic Illustration of Quantifiable Nuclear Features Illustration Features

Description

Sphericity

Sphericity of the nucleus

Fre_low_fft

Nuclear membrane regularity and smoothness

Fractal2_area

Chromatin clumping

Low_vs_high DNA

Dispersion of chromatin condensation

Gray_level_mean

Haphazard chromatin dispersion

Run_length_mean

Random distribution of chromatin

texture using local differences integrated over the object at multiple resolutions. Run-length texture features described chromatin distribution in terms of the length of consecutive pixels with the same compressed gray-level value along different orientations (0°, 45°, 90°, 135°). To make the runlength features rotationally invariant, for each run-length feature, we only used the mean and standard deviation over the four directions. 158 Am J Clin Pathol 2015;144:151-164 DOI: 10.1309/AJCPK0Y1MMFSJDKU

Low Value

Medium Value

High Value

Statistical Analysis The comparison of the value of nuclear features between the groups was performed by Wilks l (analysis of variance) to test whether there were differences between the means of at least two identified groups in a combination of dependent variables (Statistica; StatSoft, Tulsa, OK). A two-sided P value less than .05 was considered statistically significant.



A 0.60

B

0.58 0.56 0.54 0.52 0.50 0.48 0.46 0.44 0.42 0.40

C

Negative

LGD

HGD

IMC

INV

140 130 120 110 100 90 80 70 60 50 40 30 20

3.7

D 1,300

3.6

1,200

LGD

HGD

IMC

INV

Negative

LGD

HGD

IMC

INV

1,100

3.5

1,000

3.4

900

3.3

800

3.2

700

3.1

600

3.0

500

2.9

Negative

Negative

LGD

HGD

IMC

INV

400

❚Figure 1❚ Correlation of nuclear features with dysplasia progression in Barrett esophagus. A and B, Morphologic features: Sphericity measures the roundness of the nucleus shape (A); Fre_low_fft measures the regularity of the nuclear membrane (B). C and D, Chromatin features: low_vs_high DNA measures the dispersion of the chromatin at a different level of condensation inside the nucleus (C); Fractal2_area measures the presence of marked chromatin clumps (D) (P < .00001). HGD, high-grade dysplasia; IMC, intramucosal adenocarcinoma; INV, invasive adenocarcinoma; LGD, low-grade dysplasia; Dot, mean; vertical bar, 95% confidence intervals.

Results Quantification of Nuclear Changes From Normal Epithelium to INV Overall, a total of 8,344 nuclei were selected from the 61 samples. Nuclear feature measurements were performed on digital images according to computations and were divided into two main categories: morphologic and texture features. The list of the total nuclear features is displayed in Table 1, and the illustrations of representative features are presented in ❚Table 2❚. The morphologic features describe the nuclear size, shape, and boundary irregularities. They serve to characterize increases in nuclear size, loss of nuclear elongation, and the distortions in nuclear shape that are associated with progression of dysplastic changes in BE. Sphericity, a feature that measures the loss of nuclear elongation,


showed a significant increase as the BE lesions progressed from nondysplastic to LGD-HGD-IMC-INV ❚Figure 1A❚. In addition, abrupt angular variations in the nuclear contour, as well as any appearance of prominences or raggedness of nuclear borders, may be observed and quantified; Fre_low_ fft, which measures the regularity of the nuclear membrane, showed a significant decrease as the BE lesions progressed ❚Figure 1B❚. On the other hand, the texture features describe the variations in optical intensity over the nuclear image and present an objective and quantitative method for characterization of changes in chromatin appearance. Low_vs_high DNA, a feature that measures the dispersion of the chromatin at a different level of condensation, increased as the BE lesions progressed from nondysplasia to dysplasia to INV ❚Figure 1C❚. Fractal2_area, which measures the degree of



A

B

0.48 0.47

1.8 1.7

0.46

1.6

0.45 1.5 0.44 1.4

0.43

1.3

0.42 0.41

C

Negative

IND

D

60,000 55,000 50,000 45,000 40,000 35,000 30,000 25,000 20,000 15,000 10,000 5,000

1.2

LGD

Negative

IND

LGD

7,000 6,500 6,000 5,500 5,000 4,500 4,000 3,500 3,000 2,500 2,000 1,500 1,000

Negative

IND

LGD

Negative

IND

LGD

❚Figure 2❚ Quantitative digital pathology for the differential diagnosis at the lower end of the dysplasia spectrum in Barrett esophagus. A and B, Morphologic features: Sphericity measures the roundness of the nucleus shape (A); Harmon01_fft measures the smoothness of the nuclear membrane (B). C and D, Chromatin features: gray_level_mean measures the average level of DNA content (C); run_length_mean measures the intensity randomized structure of chromatin distribution (D). (P < .00001). IND, indefinite for dysplasia; LGD, low-grade dysplasia; dot, mean; vertical bar, 95% confidence intervals.

marked chromatin clumps, increased as the BE lesions progressed from nondysplasia to dysplasia to INV ❚Figure 1D❚. Quantification of the Differential Diagnosis in the Lower End of the BE Dysplasia Spectrum We then evaluated the contribution of QTP in discriminating among negative for dysplasia, IND, and LGD, where the rate of inter- and intraobserver variation is the highest.17,30 As shown in ❚Figure 2❚, there was a linear correlation of morphometric and chromatin texture features, increasing proportionally from negative to IND to LGD for Sphericity (Figure 2A), gray_level_mean (Figure 2C), and run_length_mean (Figure 2D) and decreasing inversely proportional for Harmon01_fft (a feature that measures the nuclear membrane smoothness) (Figure 2B). Moreover, the differences in two chromatin texture features, gray_level_mean (Figure 2C) and run_length_mean (Figure 2D), were statistically significant between the lower end BE class diagnosis (P < .0001).


Quantification of the Progressive Potential of LGD in BE Furthermore, we evaluated the contribution of selected chromatin texture features in discriminating between isolated LGD and LGD that was associated with and may have progressed into higher grade lesions. As shown in ❚Figure 3❚, there was a clear distinction between these two subgroups according to the level of the gray_level_mean feature (Figure 3A) and run_length_mean feature (Figure 3B). The mean and distribution of each of these features were consistently and significantly lower when LGD was associated with HGD, IMC, or INV (P < .00001).

Discussion Gastroenterologists treating patients with BE face a major clinical challenge as they try to identify which patients with BE are truly at risk for developing carcinoma vs which



A

B

70,000

400

60,000

350 300

50,000

250

40,000

200

30,000

150

20,000

100

10,000

50 0

0

–50

–10,000 –20,000

–100 Isolated LGD

LGD With HGD

LGD With IMC

–150

LGD With INV

C

Isolated LGD

LGD With HGD

LGD With IMC

LGD With INV

D 1.2E5

12,000

1E5

10,000

80,000

8,000

60,000

6,000

40,000

4,000

20,000

2,000

0

0

–20,000

Isolated LGD

LGD With Higher Grades

–2,000

Isolated LGD

LGD With Higher Grades

❚Figure 3❚ Quantitative digital pathology for the revelation of progressive low-grade dysplasia in Barrett esophagus. A and B, Chromatic features: gray_level_mean measures the spatial disorganization of chromatin (A); run_length_mean measures the intensity randomized structure of chromatin distribution (B) (P < .00001). Dot, mean; vertical bar, 95% confidence intervals. Box plot of the two comparative groups: low-grade dysplasia alone (C) vs low-grade dysplasia with concomitant higher grade lesions (D). Error bar, 5th and 95th percentiles; box, central cross, 50th percentile and median level; red cross, mean. HGD, high-grade dysplasia; IMC, intramucosal adenocarcinoma; INV, invasive adenocarcinoma; LGD, low-grade dysplasia.

patients may have more indolent versions of the disease that will not progress. Inclusion of histologic classification into the risk assessment of adenocarcinoma arising from BE has placed surgical pathologists at the center of clinical care and research endeavors. New diagnostic approaches to evaluate esophageal biopsy specimens from patients with BE are needed that can unlock more cellular and molecular information from biopsy specimens and provide actionable information to physicians managing patient care. Related technologies that enable the quantitative assessment of


nuclei and tissue architecture have recently been established as viable prognostic and diagnostic tools.34 This, in return, will generate a new breed of pathologists who have the means to improve early cancer detection programs and lead the paradigm shift of cancer screening.35 A reference study pointed out the potential of computerized nuclear morphometric quantification to determine the grade of dysplasia in BE as well as predict the progression to INV in patients with HGD.35 We describe in the present study similar findings using a different approach



and independent QDP system that measures 110 nuclear features, morphometric analysis to assess the nuclear shape and boundaries, and texture analysis to assess the chromatin structure and organization. Moreover, our results demonstrate that QDP can detect serial nuclear changes in the sequential progression of BE from normal epithelium to dysplastic epithelium to frank INV. We previously observed similar findings in multiple human epithelium sites, including the oral cavity,21 lung,32 cervix,36 and breast,37 while other independent groups have reported similar evidence in skin,38 colon,39 endometrium,40 and ovary.41 We have then showed that measurement of nuclear chromatin texture is significant because such changes are an indication of genetic or epigenetic changes that lead toward malignant transformation.21,32 This is also consistent with a recent study that pointed out the superiority of nuclear texture analysis over ploidy measurement in the differentiation of dysplastic from nondysplastic BE.42 It is well documented that detection of LGD in patients with BE on index endoscopy is associated with a high incidence rate of adenocarcinoma compared with patients with BE who do not have dysplasia.5 However, there is considerable variability in the interpretation of BE-associated dysplasia, not only between community and academic pathologists but also between expert GI pathologists.16,17,30,43 Less subjective markers are needed to evaluate the degree of dysplasia. Our results suggest that quantitative measurement of chromatin texture features has a better correlation with the class of BE dysplasia. As opposed to morphologic features measuring changes in nuclear size and shape, chromatin texture features are less sensitive to sectioning variation and could have a contribution in the differential diagnosis of BE classification, especially at the lower end of the dysplasia spectrum (negative vs IND vs LGD), where the poorest interobserver agreement is noted. We believe that repeatability of such findings with different QDP platforms could encourage the development of robust clinical validation studies in the future to explore a potential clinical consideration in the modern era of digital pathology. More interestingly, our study showed that it would be possible to distinguish isolated LGD vs LGD associated with concomitant higher grade lesions (HGD, IMC, INC) from random BE biopsy tissues through the detection of a differential pattern in their chromatin texture with QDP analyses. This observation correlates with the so-called theory of malignancy-associated changes (MAC), which is classically defined by the presence of subtle morphologic changes in the nuclei of nontumoral cells found in the vicinity of the malignant growth.44,45 The mechanism of MAC is not well established; however, studies have postulated that growth factors or cytokines excreted from cancer cells and released in the cellular environment may affect the surrounding


normal cells and, thus, explain the associated changes.44 By objectively assessing the chromatin texture, QDP opens the way to a more reproducible and sophisticated approach for MAC detection, thus enabling useful clinical applications in cancer screening. In this study, we found that QDP has the potential to detect LGD adjacently located to higher grade lesions (HGD, IMC, and/or INV). Since the foci of HGD or carcinoma are heterogeneously distributed over the BE mucosa and can be missed by random biopsies, it is of high interest to develop tools for reflex detection of concomitant HGD or carcinoma when making the diagnosis of LGD in a biopsy specimen. In conclusion, this study, albeit small and without prospective follow-up, is in concordance with the previous observations on the value of QDP in BE-associated dysplasia assessment and opens a new path of investigation for early esophageal cancer detection. Advanced discriminative analyses are required and will be performed by the authors for validation. Larger, multicenter studies will be required to confirm our findings in BE-related dysplasia. In the rapidly emerging era of digital pathology, QDP would undoubtedly be an asset for the pathologists to meet the highest standard of care. Improved diagnostic and prognostic testing in BE will lead to improved patient outcomes, including reductions in the incidence of esophageal cancer. In addition, better risk stratification tools will lead to more efficient endoscopic surveillance, since patients identified as high risk will proceed to available treatment such as radiofrequency ablation, while patients identified as low risk will be spared unnecessary surveillance endoscopies. Ultimately, these improvements in the efficiency of endoscopic surveillance will also affect the total cost of care for this patient population. Corresponding author: E. Celia Marginean, MD, FRCPC, FCAP, University of Ottawa, The Ottawa Hospital, CCW—Room 4251, 501 Smyth Rd, Ottawa, ON K1H 8L6, Canada; cmarginean@toh. on.ca.

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