Encapsulated papillary carcinoma of the breast: does it have a native basement membrane?

Encapsulated papillary carcinoma (EPC) is surrounded by a thick fibrous capsule‐like structure, which is interpreted as a thickened basement membrane (BM). This study aimed to describe the geometric characteristics of the EPC capsule and to refine whether it is an expansion of the BM or a stromal reactive process.


Introduction
Papillary lesions of the breast are a heterogeneous group of tumours characterised morphologically by the presence of fibrovascular cores covered by epithelial cells ranging from benign to malignant. 1 Papillary carcinomas (PCs) include encapsulated papillary carcinoma (EPC), solid papillary carcinoma (SPC), papillary ductal carcinoma in situ (DCIS), and invasive papillary carcinoma. 1,2 EPC typically present as well-circumscribed and encapsulated mass(s) that lack a myoepithelial cell (MEC) layer at their periphery in 80% of cases. 3 EPC is a term that has been introduced to define the previously called intracystic or encysted variant of PC to reflect its encapsulation. 3 EPC exhibits a peripheral thick capsule-like structure, which was interpreted as a thickened basement membrane (BM) as it contains some of the BM structures, namely laminin and collagen type IV. Subsequently, this feature was used as one of the indications of its in situ nature. 4 BM is a thin, pliable sheet-like material of extracellular matrix (ECM) that sits between normal or proliferative intraductal epithelial and MEC and the surrounding connective tissue. 5 In malignant tumours, preservation of BM denotes in situ disease, while its absence is a feature of invasive tumours. 5 Although some DCIS shows expansion of the BM, this is different from the reactive fibrous tissue capsule seen around some benign and malignant tumours in various tissue and organs including renal cell carcinomas, hepatocellular carcinomas and thyroid follicular carcinomas, and their benign counterparts. 6 Tumour encapsulation refers to the presence of a fibrous capsule surrounding the neoplasm and is often associated with indolent clinical behaviour. 6 The concept of capsule pathogenesis suggests that tumour encapsulation is not a passive condensation of fibrous tissue by expansive growth or a body defence mechanism against tumours as a foreign body response, but is a biological process resembling wound-healing responses. 6,7 The World Health Organization (WHO) recommend that EPC are staged as carcinoma in situ (pTis) in cases where there is an absence of frank stromal invasion or infiltrative growth despite the lack of an MEC layer. 8 This was supported by some previous studies showing expression of some BM structures, such as laminin and collagen type IV in EPC, and inferred that the structure around EPC represents expansion of the BM, and hence the intraductal nature of the tumour. 1,9 The outer layer of the BM that faces the ECM is called the reticular lamina, or lamina fibro-reticularis, and is mainly formed of collagen III. 5 Previous studies have demonstrated that collagen III coexpressed with collagen I, resulting in regulation of fibrils dimensions that influence their mechanical stiffness and functional properties. 10,11 Collagen III has a key role in normal tension maintenance and modulating scar formation. 10 In addition, BM stiffening has been reported to trigger cell invasiveness in prostate cancer 12 and squamous cell carcinoma. 13 Picrosirius red (PSR) stain is a special histochemical stain for collagen. PSR has a higher specificity for collagen fibre detection compared to other commonly used stains, as it can stain finer collagen fibrils, which in turn avoids underestimation of collagen density. 14 PSR is comprised of elongated birefringent dye molecules that can bind to the collagen fibres in parallel positions to the fibres, thus improving the natural birefringence under a polarizing microscope. It therefore detects thinner fibres (type III collagen) that are visualised as green to greenish yellow colours, while thicker fibres (type I collagen) present as yellowish orange to orange red colours. 15,16 Therefore, the aim of this study was to ascertain whether the EPC capsule represents a reactive process similar to that seen in some invasive tumours, or an expansion of the native BM mimicking that is seen in some cases of DCIS, and hence explain the intraductal nature of the tumour on the basis of BM geometric characteristics.

S T U D Y C O H O R T
This study included 100 cases of normal and neoplastic breast tissue from patients who presented to Nottingham City Hospital, Nottingham, UK between 1999 and 2006. The cases were divided into four groups, each containing 25 patients. The four groups included encapsulated papillary carcinoma (EPC), in addition to ductal carcinoma in situ (DCIS), normal breast tissue, and invasive carcinoma (INV) as control groups. The INV group included adenoid cystic carcinoma, invasive cribriform, and salivary gland-like carcinoma, as these meet the criteria of being aggressive tumours and showed BM-like material surrounding the neoplastic cells. Encapsulated papillary thyroid carcinoma (EPTC) cases were also stained and analysed for comparison against the EPC capsule group and used as a control group.

T I S S U E P R O C E S S I N G A N D S T A I N I N G
Freshly cut, 4-lm thick, full-face sections for the cohort were stained with haematoxylin and eosin (H&E) or picrosirius red (PSR) special stain; one section per case from each tumour block was prepared H&E-stained slides were examined, via standard light microscopy with a 109 objective, for histopathological verification of the section. The glands were identified that served as a reference once the tissue sections had been stained with PSR.
The PSR-stained tissue was examined under polarised microscope and visualised using the accompanying software (Leica, IL 5000B, and LAS VER-SION 3.8.0, Leica Microsystems, Switzerland) and a camera. Under polarised light, PSR-stained collagen type I presents within the red-orange-yellow range and type III as green; under light microscopy, total collagen can be visualised within the red spectrum and the two collagen types cannot be differentiated. 17 Tissue sections were split into four quadrants. From each quadrant a single gland was chosen, and systematic, nonoverlapping, random areas were examined in a Z shaped pattern; 4-6 photomicrographs were captured. Each gland was further split into four quadrants; two regions of interest (ROIs; 75 9 75 pixels) were randomly selected per quadrant and two measurements were taken from each quadrant with one measurement per ROI (eight measurements per gland, 32 measurements per slide). This method was validated by repeating the calculation the mean using different randomly selected measurements (always n = 32 measurements), with no significant differences in the mean values obtained. The mean values of the measurements were calculated for each case. All photomicrographs were taken at 1009 magnification.
The EPC group was further analysed to assess the difference between inner and outer parts of the thickened capsule. As the BM of normal and DCIS was thin, such splitting into inner and outer was not possible and the whole BM was analysed as one part and the difference was considered between different ROIs. The inner part of the capsule was defined as the most adjacent part to tumour cells, while the outer capsule part was defined as the fibrous part adjacent to the stroma.

I M A G E A N A L Y S I S A N D Q U A N T I F I C A T I O N O F
C O L L A G E N Assessment of BM thickness and collagen distribution was undertaken using ImageJ (NIH, Bethesda, MD, USA, available for free download at https://imagej. nih.gov/ij/download.html).

Basement membrane thickness
Thickness of the BM and the EPC capsule was measured by calculating the average of 20 measurements for each case at 209 magnification.

Proportion of thin and thick fibres
The collagen fibres were distinguished as either thin or thick fibres using the polarised light, thin fibres were greenish yellow, and thick fibres appeared orange red. 18 An ImageJ macro plugin recorder was used to automate channel separation for red, green, and background colours with quantification of the pixel area for each feature. Using the raw data output, the proportions and mean values of thin and thick collagen fibres were calculated for each case.

Collagen density
Assessment of collagen density was conducted via ImageJ split into RBG channels (red, blue, green); the blue and red images were then excluded. The green image was adjusted into a binary image with red foreground pixels designated as intensity 0 and the background was expressed as white pixels (intensity 255). All images were adjusted to a threshold of 150 to decrease noise. Integrated density was measured in pixels using the automated tools in ImageJ. The mean values for each case were calculated.

Collagen length, straightness, and width
Assessment of collagen fibre features using CT-FIRE (ctFIRE_V2.0Beta_WIN64_MCR2014b.exe) was conducted to extract individual fibres and to quantify the following measurements (units = pixels): mean fibre length-defined as the total contour of the fibres; mean fibre straightness-defined as the end-to-end length; and mean fibre width. The values for each of the fibres, from each image, for each tumour, were averaged to determine one value for each case.

Collagen fibre alignment and orientation
Assessment of collagen fibre features using Curve Align (CurveAlign_V4.0Beta_WIN64_MCR2014b.exe) was undertaken to extract individual fibres, and then quantify the following measurements: the alignment coefficients for each fibre, ranging from 0 to 1 for each fibre, with 0 used to designate random alignment and 1 assigned as perfect alignment (alignment of fibres to each other); mean orientation angle (expressed as degrees ranging from 0 to 180°) that was calculated and defined as the alignment of each fibre to a horizontal line.

S T A T I S T I C A L A N A L Y S I S
For all statistical analysis, P < 0.05 was considered statistically significant and undertaken either in SPSSv26 (IBM, Armonk, NY, USA) or GraphPad Prism (Boston, MA, USA). Kruskal-Wallis, one-way analysis of variance (ANOVA), chi-square test, paired T test, and Wilcoxon signed ranks tests were used as specified in each result. Graphs and tables were constructed as applicable using GraphPad Prism. Results are expressed as the mean AE SD of the mean throughout unless otherwise stated.

C A P S U L E A N D B M T H I C K N E S S A N D C O L L A G E N D I S T R I B U T I O N
A significant increase in the thickness of the EPC capsule (59.65 AE 18.50 lm) was found in comparison to the BMs in the normal breast tissue group (0.032 AE 0.009 lm; P < 0.0001) and BMs from DCIS cases (0.058 AE 0.017 lm; P < 0.0001) (Figure 1). In contrast, there was no difference between the BM thicknesses in the DCIS samples (0.058 AE 0.017 lm) and normal breast tissue (0.032 AE 0.009 lm; P < 0.995; Figure 1).
Regarding the fibre types, most of the EPC capsule collagen was comprised of type I (75. 46  There was no statistically significant difference between BM of the normal breast tissue and those in DCIS regarding thickness (P < 0.995) or collagen distribution (P < 0.07). Contrary to this, there was a significant increase of collagen density in DCIS cases (P < 0.0001).

C O L L A G E N F I B R E C H A R A C T E R I S T I C S
The EPC capsules showed significant increases in fibre width (7.78 AE 0.15 pixels, P < 0.0001) and decreases in length (56.53 AE 2.74 pixels, P < 0.0001) compared to both normal and DCIS BM width (7.05 AE 0.15 and 7.07 AE 0.2 pixels, respectively) and length (61.66 AE 2.78 and 61.61 AE 3.89 pixels, respectively). EPC capsules also had straighter fibres (0.95 AE 0.004 on a 0-1 scale) compared to BMs in both normal breast tissue (0.92 AE 0.007 on a 0-1 scale) and DCIS (0.93 AE 0.005 on a 0-1 scale, P < 0.0001). No significant differences were observed between normal and DCIS BMs in either fibre width (P < 0.96) or length (P < 1); however, DCIS BM fibres were considered more straight in formation compared to those in normal BMs (P < 0.0001; Table 1, Figure 4).
EPC capsule fibres presented with less alignment (0.34 AE 0.07 on a 0-1 scale) compared to normal and DCIS BMs (0.57 AE 0.08 and 0.40 AE 0.09, respectively, on a 0-1 scale, P < 0.0001, Table 1, Figure 4). There was also a decrease in fibre alignment in the DCIS BM compared to normal BMs (P < 0.0001; Table 1, Figure 5).
The orientation angles of the collagen fibres were classified as either 'parallel' or 'perpendicular' for each case observed based on the dominant angle measurement. Angles of 70-110°were considered 'perpendicular' fibres and measurements outside of this were considered 'parallel'. Comparisons were made between the numbers of cases in each group classified as having either 'parallel' or 'perpendicular' fibres in the BM. Most of the cases within the EPC group showed more 'perpendicular' fibre (96%) compared to DCIS (60%), whereas no cases in the normal tissue had perpendicular fibres in their BMs. Conversely, 4% of EPC cases had 'parallel' fibres compared to 40% of DCIS cases. All cases of normal breast tissue group had 'parallel' fibres (P < 0.0001, Table 2, Figure 5).
There was a significant increase in collagen fibre density in EPC capsule cases (53.47 AE 4.27 pixels) compared to the invasive group BM-like material (38.90 AE 6.46 pixels, P < 0.0001), with no difference in collagen fibre content for either collagen I (P < 0.86) or collagen III (P < 0.37). In addition, EPC capsule fibres showed a significant increase in length and straightness (both P < 0.0001) compared to invasive fibres, while there were no significant differences in fibre width between both groups (P < 0.98). With regard to collagen fibre directions, EPC cases showed more aligned fibres compared to the invasive group (P < 0.0001; Table 1).

C O M P A R I S O N B E T W E E N E P C C A P S U L E A N D E N C A P S U L A T E D P A P I L L A R Y T H Y R O I D C A R C I N O M A ( E P T C ) C A P S U L E
There was no significant difference between the EPC and EPTC capsule thickness (59.65 AE 18.50 and 57.76 AE 13.86 lm, respectively, P = 0.77; Figure 6) or fibre density (53.47 AE 4.27 and 55.29 AE 3.74 pixels, respectively, P = 0.25).  Figures 7  and 8). There were also no significant differences between EPC capsule fibre width (7.78 AE 0.15 pixels) and length (56.53 AE 2.74 pixels) compared to EPTC capsule fibre width (7.61 AE 0.35 pixels; P = 0.06) and length (54.85 AE 3.59 pixels P = 0.14).
In contrast, the EPC capsules did present with straighter fibres (0.95 AE 0.004 on a 0-1 scale) compared to those within the ETPC capsule (0.94 AE 0.005 on a 0-1 scale; P < 0.034). In terms of directionality, the EPC capsule fibres presented with less alignment (0.34 AE 0.07 on a 0-1 scale) compared to those in the  EPTC capsules (0.38 AE 0.04 on a 0-1 scale); however, the difference was not significant (P = 0.12). 96% of EPC (96%) had 'perpendicular' fibre compared to 80% of EPTC but the difference was not significant (Figure 8).

H E T E R O G E N E I T Y O F T H E E P C C A P S U L E
Comparisons of measurements between the inner and outer parts of the capsule tissue were also undertaken in the EPC group. This showed that the outer part of the capsule had significantly higher collagen density (42.50 AE 1.78 pixels; paired t-test, t = 5.88,    Figures 9 and 10). There was a statistically significant increase in mean fibre width in the outer part of the capsule (7.78 AE 0.015 pixels) compared to the inner part of the capsule (7.36 AE 0.094 pixels; paired t test, t = À11.90, P < 0.0001). There were no significant differences in mean fibre lengths within both the inner and outer capsule (57.59 AE 1.62 compared to 57.05 AE 1.99 pixels, respectively; t = À1.35, P < 0.19). The outer capsule fibres showed more straightness (0.95 AE 0.005 on a scale of 0-1) compared to the inner part (0.94 AE 0.007; Wilcoxon signed ranks test, Z = -4.32, P < 0.0001). Capsular collagen fibres of the outer part were also less aligned than the inner part (t = À6.07, P < 0.0001; Figure 10).

C O M P A R I S O N B E T W E E N E P C C A P S U L A R H E T E R O G E N E I T Y A N D O T H E R G R O U P S
Both EPC capsular parts inner (40.47 AE 1.31 pixels) and outer (42.50 AE 1.78 pixels) showed increased collagen density (P < 0.0001) compared to other groups (normal, DCIS, and invasive), and showed higher collagen I content and lower collagen III compared to other groups except the invasive group (Table 3). In contrast, there was no significant difference in both collagen I and III (both P < 0.38) content between the outer part of the capsule and invasive groups. Both the inner and outer parts showed increases in fibre width, length, and straightness compared to other groups (all P < 0.0001), while the outer part showed no significant difference in width compared to the invasive group (P < 0.97) and the inner part showed no significant difference regarding alignment (P < 1) compared to the invasive group, or straightness with both DCIS and invasive (P < 0.13 and P < 0.94, respectively). These results are shown in Table 3 and Figure 11.

Discussion
PC remains a controversial entity regarding morphological features, histological categorization, and clinical management. Although the biological behaviour of papillary DCIS and invasive PC is well established, the categorization of EPC into in situ or invasive disease remains challenging and problematic, resulting in management implications as categorizing as an invasive lesion may trigger adjuvant systematic therapy. 19 The current study results showed that the BM that surround the normal ducts and lobules in normal breast tissue had predominantly collagen type III fibres, with thinner, shorter, and more curved fibres, which were parallel to the duct border and showed good alignment to each other. The DCIS BMs were similar to normal breast tissue but exhibited higher density and less aligned straighter fibres. However, the BM-like material that surrounded the invasive group tissue showed a high density of collagen fibres with increased proportions of collagen I and decreased collagen III, with thicker, shorter, straighter, and less aligned fibres.
A study of EPC showed that the presence of collagen IV and laminin in all cases of pure EPCs with an absence of their expression in a significant percentage of EPCs associated with invasive breast cancer, and these results were used to indicate that these collagen IV and laminin-positive tumours may be characterised as in situ lesions. 1 However, laminin and collagen IV can be seen in the wound-healing process, and therefore not specific for the native BM characteristic of in-situ tumours. 20,21 Moreover, a similar peripheral capsule-like structure can be seen surrounding invasive tumour foci in other tissues outside the breast, supporting the theory that this is a reactive process rather than BM expansion. 4 In addition, some cases of typical EPC with a well-developed capsule have been reported with local muscle infiltration, 22 lymph node metastasis, [22][23][24] or distant Figure 6. Capsule thickness. Measurements were obtained on picrosirius PSR-stained histology specimens at 209 magnification, Ima-geJ. 20 measurements were obtained for each case. EPC, encapsulated papillary carcinoma capsule; EPTC, Encapsulated papillary thyroid carcinoma, Independent t test t = 0.881, P < 0.77. metastases 25-27 and a similar capsule-like structure is seen around some of the EPC foci at the distant sites. 22 These findings argue against the surrounding capsule of EPC that lack MEC being a native but thickened BM characteristic of the intraductal lesions and provides further evidence that these lesions are invasive carcinomas with an indolent behaviour and expansile growth pattern. 3 Not all sheet like structures at the epithelial stroma interface represent BM. A true capsule is defined as a rim of relatively pure fibrous tissue (composed predominantly of collagen) around a tumour, while a pseudocapsule is defined as mixed fibrous tissue and entrapped normal structures around the tumour. A pseudocapsule is mainly caused by the expansive growth of the tumour and a combination of the mechanical effect of the compression of the surrounding tissue and condensation of scarring that occurs as a body defence mechanism against tumours. Meanwhile, a true capsule is a biological process resembling wound-healing responses and has morphological integrity. Many tumours, including benign and malignant, in situ, and invasive, exhibit an encapsulation process; however, the distinction between true and pseudocapsules varies based on the context. 6 The present study compared the characteristics of collagen fibres in EPC capsules with the BMs of normal and DCIS, as well as with the BM-like material that is present in some invasive breast tumours. This showed that EPCs had a thickened capsule with denser collagen fibres, enriched with stromal-type collagen (collagen I) with thicker, shorter, straighter, and more disorganised fibres than that of the BM of DCIS or normal breast tissue. The present study also demonstrated that the EPC capsule exhibited no significant difference to the BM-like material surrounding the invasive types regarding many characteristics such as collagen I and collagen III fibre content, and fibre width. In addition, EPC capsules showed marked heterogeneity between their inner and outer parts, with the inner part resembling the DCIS BM only in fibre straightness; however, there was a significant increase in the capsule thickness.
The outer part of EPC capsules exhibited no significant differences relating to fibre density, width, or collagen distribution (I and III) compared to the invasive tissue.
A previous study showed that EPC lesions express higher levels of TGFb1 compared with DCIS and invasive carcinomas, which plays a role in the development of the thick fibrous capsule, supporting the hypothesis that the EPC capsule is a reactive rather than a thickened expanded native BM resulting from distention by the proliferation of neoplastic papillary. 3 These observations were consistent with the results from the present study. All of the DCIS, EPC capsule (inner and outer), and invasive group cases had fewer thin fibres compared to the control group. In addition, this study showed that most of the EPC capsule collagen was type I compared to BMs from both normal breast tissue and DCIS, with no difference exhibited when compared with the BM-like material in invasive tumours.
Many previous studies focused on the distribution of collagen IV around EPC to examine whether this tumour was in situ or invasive. Esposito et al. concluded that most cases of EPC were surrounded by collagen IV. 9 Wynveen et al. identified continuous, high intense layers (3+ positivity) of collagen IV in three cases; however, most cases had discontinuous and fragmented (1+, 2+) expression and only one case exhibited no collagen IV reactivity. 28 Golestani et al. demonstrated the presence of continuous, uniform, and strong collagen IV immunostaining around all EPC cases. 29 In addition, Akladois et al. documented the presence of BM around EPC by positive collagen IV staining. 30 However, the presence of BM components surrounding EPCs cannot be considered as evidence for in situ carcinomas, as this finding has been detected in a subset of invasive breast cancers and in nodal metastases. [31][32][33] Encapsulated papillary thyroid carcinoma (EPTC), which is an indolent carcinoma with rare recurrence or lymph nodal metastases 34-38 exhibits excellent prognosis, 39,40 it is also typically surrounded by a fibrous tissue capsule similar to EPC. Quantitative analysis of collagen fibres using Second Harmonic Generation (SHG) microscopy revealed that EPTC capsules had more dispersed collagen fibres than benign tumour capsules. In line with our findings, Bayadsi et al., 41 reported that EPTC had higher amounts of collagen I within the capsule using  immunohistochemistry . Additionally, other invasive tumours with indolent behaviour characterised by pushing borders and surrounded by a fibrous capsule that contained both collagen IV and collagen I fibres, including hepatocellular carcinoma [42][43][44][45][46] and some forms of prostate adenocarcinoma. 47 This was consistent with results obtained in the present study.
Renal papillary carcinoma is an encapsulated tumour in the kidney 48 that is surrounded by a fibromuscular capsule that has a complex of collagen fibres and smooth muscle. 49 Many studies have detected collagen IV in the renal pseudocapsule. [50][51][52] However, Lohi et al., in their study of collagen type IV a1(IV)-a6(IV) expression in renal carcinoma showed that papillary the carcinoma capsule had only collagen IV alpha 5 chain. 53 Another study that characterised papillary carcinoma capsule using multiphoton microscopy showed that there was a significant difference in thickness and collagen area variation within each tumour; however, the thickest and thinnest parts showed no significant differences regarding collagen density or fibre directions. 54 In addition, when comparing papillary renal carcinoma with other types, it showed a lower rate of capsule completeness and presence. 54 Stamatiou et al. demonstrated that a renal carcinoma capsule expressed collagen I & IV, myofibroblast-like SMA-and vimentinpositive cells using immunohistochemistry, which suggested that this capsule was generated from host mesenchymal cells. 55 These findings are consistent with the present study results showing that the EPC capsule is enriched with collagen I.
In conclusion, EPC is surrounded with BM-like material resembling those surrounding some invasive tumour types and is significantly different from the BM of normal breast tissue and DCIS. This study provides further evidence that EPC is an indolent invasive carcinoma surrounded with BM-like material with biopathological features between in situ and invasive carcinoma on the basis of capsule characteristics. Most papillary tumours in different organs that are surrounded with BM-like material showed indolent behaviour with better prognosis; therefore, mechanisms that explain these phenomena should be further investigated.