Periductal Stromal Collagen Topology of Pancreatic Ductal Adenocarcinoma Differs From That of Normal and Chronic Pancreatitis

Cole R Drifka; Jo Tod; Agnes G Loeffler; Yuming Liu; Gareth J Thomas; Kevin W Eliceiri; Kao W John


Mod Pathol. 2015;28(11):1470-1480. 

In This Article


Quantification of Collagen Topology in Whole-tissue Microarray Cores

The widespread clinical application of tissue microarrays has enhanced the efficiency with which diagnostic, prognostic, and therapeutic biomarkers are screened and assessed by accounting for tumor heterogeneity through increased patient representation.[27] Pathology-reviewed clinical tissue cores were imaged in entirety using a second harmonic generation stitching algorithm, which produced submicron resolution information about collagen distribution and topology in relation to epithelial structures. Even within standard diameter cores, a wide variety of histological features were qualitatively observed (Figure 1). For example, normal ducts were sparsely distributed among variable amounts of acinar and adipose tissue. In cores of chronic pancreatitis, the extent of fibrosis around the ducts varied considerably as well. Some ducts appeared similar to normal ducts, whereas some were contorted in the context of abundant collagen and superficially resembled malignant ducts.

Figure 1.

Pancreatic tissue heterogeneity. Whole-tissue microarray cores were visualized in entirety using traditional hematoxylin and eosin (H&E) histopathology (left column) and corresponding second harmonic generation (SHG) signal (right column, shown in red). Shown are representative cores determined to be as normal pancreatic tissue chronic pancreatitis, pancreatic ductal adenocarcinoma grade 1 (PDAC-G1), pancreatic ductal adenocarcinoma grade 2 (PDAC-G2), and pancreatic ductal adenocarcinoma grade 3 (PDAC-G3). Different tissue features and patterns of fibrosis are observed within single cores. AC, acinar tissue; ADI, adipose tissue; BV, blood vessel; CP, ducts in chronic pancreatitis; G1, well-differentiated (grade 1) pancreatic ductal adenocarcinoma duct; G2=moderately differentiated (grade 2) pancreatic ductal adenocarcinoma duct; G3, poorly differentiated (grade 3) pancreatic ductal adenocarcinoma duct; N, normal duct; PNI, perineural invasion; ↑, inflammatory cell infiltration; *, connective tissue conforming to normal lobular architecture; #, highly aligned tracks of connective tissue. All images are shown at the same scale. Scale bar=250 microns.

Although pancreatic ductal adenocarcinoma tissues display an overall increase in fibrosis compared with benign tissues, we noted that the appearance of collagen was heterogeneous throughout individual cores. In some tumors, collagen appeared sparsely deposited around highly cellular regions, whereas in other specimens, highly aligned collagen structures traversed the tissue with few intervening epithelial structures. In addition, the distribution of ductal epithelium (widespread vs sparse), different histological grades of the malignant glands, and occasional presence of blood vessels, nerves, adipose tissue, and infiltrating inflammatory cells contributed to the overall tissue heterogeneity of individual tissue cores.

The structure and organization of collagen fibers were objectively assessed in whole-tissue microarray cores by quantifying the corresponding second harmonic generation images. Despite the tissue heterogeneity observed in the tissue microarray cores, collagen fibers in the pancreatic ductal adenocarcinoma-associated stroma were significantly more aligned and longer than in normal pancreatic tissue, underscoring our initial qualitative observations (Figure 2). In addition, fibers in pancreatic ductal adenocarcinoma tissue were significantly more aligned than fibers in the setting of chronic pancreatitis (P<0.001). When pancreatic ductal adenocarcinoma tissues were categorized by histological grade, a significant correlation between grade and collagen metrics was not observed (Supplementary Figure S1

Figure 2.

Quantification of collagen fiber alignment, length, straightness, and width in different pathology-reviewed whole-pancreatic tissue microarray cores. n=84 normal, 49 chronic pancreatitis (CP), 508 pancreatic ductal adenocarcinoma (PDAC) cores from 241 patients. Data bars represent adjusted means±standard error. *P<0.05, **P<0.001, ***P<0.0001.

Stromal Collagen Topology Differs Around Distinct Pancreatic Tissue Features

Although tissue microarray cores have been shown to be representative samples of the entire tumor,[28] inherent tissue heterogeneity still exists. Without considering the spatial distribution of specific tissue components, it is unclear as to where characteristic collagen changes are occurring. To address this, we analyzed collagen in the immediate vicinity of structural elements within the tissue (ducts, blood vessels, nerves) and malignant epithelium, as identified by a pathologist on hematoxylin and eosin-stained material. Figure 3 depicts qualitative collagen attributes that were observed relative to different epithelial structures. Normal ducts are comprised of a single layer of cuboidal epithelium that contact an intact basement membrane that is maintained by resident pancreatic stellate cells.[29] Separating the normal duct from the rest of the pancreatic parenchyma is a fibrotic cuff largely composed of short collagen fibers that are concentrically oriented around the duct lumen. Similar concentric collagen organization is observed in the stroma around blood vessels and nerves (Figure 4). In normal acinar tissue regions not in the immediate vicinity of ducts, collagen fibers are relatively sparse and when detected, conform to the normal lobular architecture of the pancreas. In chronic pancreatitis, ducts appear contorted and the periductal stroma is characterized by disorganized collagen fibers that interdigitate between the epithelial cells. Also, inflammatory cells are commonly observed dispersed throughout the immediately adjacent stroma. In areas of infiltrating pancreatic ductal adenocarcinoma, a significant increase in the overall density of fibers is readily apparent in all histological grades. The collagen of well-differentiated (grade 1) malignant ducts, which commonly resemble benign ducts if only epithelial atypia is considered, is strikingly more elongated and aligned in the immediate vicinity of the pancreatic ductal adenocarcinoma cells. Around moderately differentiated (grade 2) or poorly differentiated (grade 3) pancreatic ductal adenocarcinoma ducts, a similar collagen phenotype is observed. In our survey of 628 distinct pancreatic ductal adenocarcinoma regions, we saw that these collagen features can be either widespread or detected only at discrete foci around the epithelium. Interestingly, collagen also appeared reorganized at the infiltrating front of pancreatic ductal adenocarcinoma tumors, indicating a potential involvement of collagen reorganization in cancer cell invasion (Figure 4).

Figure 3.

Key histological features of different representative stroma–epithelial interfaces in pancreatic tissue visualized using traditional hematoxylin and eosin (H&E) histopathology (top row) and corresponding second harmonic generation (SHG) signal (bottom row, shown in red). Normal ducts and benign ducts in chronic pancreatitis display a loose, concentric organization of collagen fibers around the epithelium. In pancreatic ductal adenocarcinoma tissues of all grades, cancer cells are surrounded by an ordered periductal stroma characterized by focally aligned and elongated collagen fibers. All images are shown at the same scale. Scale=100 microns.

Figure 4.

Key features of different tissue hallmarks in pancreatic tissue visualized using traditional histopathology (top row) and corresponding second harmonic generation (SHG) signal (bottom row, shown in red). First and second columns: normal blood vessels and nerves display a concentric organization of collagen fibers relative to the vessel lumen and nerve body, respectively. Third column: the front of a pancreatic ductal adenocarcinoma tumor infiltrating into the duodenal muscularis externa is characterized by distinct aligned and elongated collagen fibers orientated in the direction of invasion. Last column: immunohistochemical staining for alpha-smooth muscle actin (αSMA, brown) in pancreatic ductal adenocarcinoma tissue displaying the distribution cells relative to aligned and elongated collagen fibers running parallel to each side of a ductal protrusion. All images are shown at the same scale. Scale=100 microns.

As collagen features appeared qualitatively distinct around ducts in three histologically distinct tissue types, we sought to determine whether quantitative parameters analyzed with CT-FIRE could distinguish whether a duct is normal, present in chronic pancreatitis, or malignant. Consistent with our whole-core analysis, the stroma of pancreatic ductal adenocarcinoma ducts showed statistically significant increases in collagen fiber alignment and length compared with normal and chronic pancreatitis stroma regardless of histological grade (Figure 5, Supplementary Figure S2 By focusing the analysis to the immediate vicinity of the epithelium rather than the entire stroma of the tissue core, we were able to achieve greater statistical power than with whole-core analysis. This underscores the inherent issue of tumor tissue heterogeneity and the need to account for the spatial distribution of cancer cells within the fibrotic stroma, and in relation to other, non-malignant cell types present. Furthermore, we saw that the pancreatic ductal adenocarcinoma stroma has collagen fibers that are significantly straighter and thicker, a finding that was not readily apparent in whole-core analysis. To determine which collagen fiber features are most powerful in discriminating duct types, we evaluated receiver operating characteristic curves. As shown in Figure 6 and Table 1 and Table 2, a statistically significant difference was demonstrated between the pancreatic ductal adenocarcinoma and benign regions. Of the four collagen metrics, collagen fiber length had the most value in differentiating the regions with an area under the curve of 0.733 for pancreatic ductal adenocarcinoma vs benign and 0.701 for pancreatic ductal adenocarcinoma vs chronic pancreatitis. These findings indicate that a unique collagen topology can be detected in the pancreatic ductal adenocarcinoma stromal microenvironment on the basis of second harmonic generation imaging and quantification compared to normal pancreas and chronic pancreatitis.

Figure 5.

Quantification of collagen fiber alignment, length, straightness, and width in different pathologist-annotated periductal stroma types. n=67 normal, 71 chronic pancreatitis (CP), 628 pancreatic ductal adenocarcinoma (PDAC) regions from 117 patients. Data bars represent adjusted means±standard error. *P<0.05, ***P<0.0001.

Figure 6.

Receiver operating characteristic curves for differentiating stroma types based on collagen fiber features extracted by CT-FIRE. Left: pancreatic ductal adenocarcinoma (PDAC) regions vs benign regions (chronic pancreatitis and normal ducts). Right: pancreatic ductal adenocarcinoma regions vs only chronic pancreatitis (CP) regions. The line of no effect (area under the curve=0.500) is shown as a dashed black line. n=67 normal, 71 chronic pancreatitis, 628 pancreatic ductal adenocarcinoma regions from 117 patients.