Pathology – Research and Practice 210 (2014) 879–884

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Original Article

Collagen, type XI, alpha 1: An accurate marker for differential diagnosis of breast carcinoma invasiveness in core needle biopsies Javier Freire a,b,∗ , Saioa Domínguez-Hormaetxe d , Saray Pereda a,b,c , Ana De Juan e , Alfonso Vega f , Laureano Simón d , Javier Gómez-Román a,b,c a

Hospital Universitario Marqués de Valdecilla, Anatomía Patológica, Santander, Spain IDIVAL, Spain c Cantabria University, Spain d Oncomatryx SL, Bilbao, Spain e Hospital Universitario Marqués de Valdecilla, Oncología médica, Santander, Spain f Hospital Universitario Marqués de Valdecilla, Radiodiagnóstico, Santander, Spain b

a r t i c l e

i n f o

Article history: Received 8 January 2014 Received in revised form 6 May 2014 Accepted 22 July 2014 Keywords: Breast cancer Infiltrating tumor differentiation Core needle biopsy Collagen, Type XI, Alpha 1

a b s t r a c t Accurate diagnosis of invasive breast lesions, when analyzed by Core Needle Biopsy, may suppose a major challenge for the pathologist. Various markers of invasiveness such as laminin, S-100 protein, P63 or calponin have been described; however, none of them is completely reliable. The use of a specific marker of the infiltrating tumor microenvironment seems vital to support the diagnosis of invasive against in situ lesions. At this point, Collagen, type XI, alpha 1 (COL11A1), might be helpful since it has been described to be associated to cancer associated fibroblasts in other tumors such as lung, pancreas or colorectal. This paper aims to analyze the role of COL11A1 as a marker of invasiveness in breast tumor lesions. Two hundred and one breast Core Needle Biopsy samples were analyzed by immunohistochemistry against pro-COL11A1. The results show a significant difference (p < 0.0001) when comparing the expression in infiltrative tumors (93%) versus immunostaining of non-invasive lesions (4%). Forty cases of underestimated DCIS were also stained for COL11A1, presenting a sensitivity of 90% when compared with p63 and calponin which not tagged invasion. In conclusion, pro-COL11A1 expression is a promising marker of invasive breast lesions, and may be included in immunohistochemical panels aiming at identifying infiltration in problematic breast lesions. © 2014 Elsevier GmbH. All rights reserved.

Introduction Breast cancer presents the highest incidence and is the second cause of deaths for neoplastic diseases among women [1]. Although mortality has been greatly reduced in the last few years, mainly due to an early detection [2], breast cancer is still a major social problem in developed countries. This is why new efforts are needed to understand this disease, leading to better diagnosis, prognosis and treatment. The epithelial-mesenchymal transition (EMT) enables epithelial cells to acquire migratory capacity, invasiveness and resistance to apoptosis [3]. In breast, this is not only a disease-associated event,

∗ Corresponding author at: Division of Anatomophatology, HUMV, Avda. Valdecilla s/n, 39008 Santander, Spain. Tel.: +34 666432446. E-mail address: [email protected] (J. Freire). http://dx.doi.org/10.1016/j.prp.2014.07.012 0344-0338/© 2014 Elsevier GmbH. All rights reserved.

since EMT has also been reported during duct morphogenesis in mammary gland [4,5]. Several neoplastic cell-associated pathways that control this process and which are associated with tumor progression and metastasis have been described [6]; however, the importance of tumor-surrounding microenvironment for EMT and malignant transformation has also been widely discussed [7–10]. The microenvironment on which the tumor grows is complex, consisting mainly of tumor epithelial cells and cancer-associated fibroblast (CAF) [11]. Several studies support the hypothesis of a cross-talk mechanism between CAFs and tumor cells. For example, the co-culture of CAFs with breast tumor cells increased their metastatic ability [11,12]. In fact, it has been demonstrated that the removal of the tumor microenvironment, and more specifically of the CAFs, reduces its invasive capacity [13]. CAFs are involved in tumorigenesis, through the synthesis, deposition and remodeling of the extracellular matrix (ECM) [12,14,15]. Collagen is the major component of the ECM, and the balance between its

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synthesis and degradation plays a vital role for physiological conditions. During tumor progression, an intensified degradation of collagen takes place, thus enabling migration of epithelial cells [16,17]. For this reason, collagen has become a key element in the fight against cancer [18–20]. Type XI collagen alpha 1 (COL11A1) is a minor component of collagen. Although it has been described in various tissues [21], it is mainly expressed in cartilage, where it helps maintaining the spacing and diameter of Type II collagen fibers [22]. Mutations of COL11A1 have been associated with Stickler [23] and Marshall syndromes [24], and other chondroid diseases such as fibrochondrogenesis [25,43]. The first description of COL11A1 in cancer was made by Kleman et al. [26] in a rhabdomyosarcoma cell line, and since then COL11A1 has been studied in different neoplasms like pancreatic [27], gastric [28], colorectal [29–31] or pulmonary [32], where it is always overexpressed when compared with its adjacent normal tissue or inflammatory lesions. In breast cancer, COL11A1 gene expression significantly increases in invasive ductal carcinoma (IDC) when compared with in situ ductal carcinoma (DCIS) [33,34], although its specific protein expression has not been yet well characterized due to the lack of antibody able to detect COL11A1 specifically. Taking into account the reports showing COL11A1 overexpression in different tumors, including breast cancer, and the lack of information about COL11A1 expression in non-neoplastic breast disease, we decided to evaluate the usefulness of a new highly specific anti pro-COL11A1 monoclonal antibody for the differential diagnosis between infiltrating and several non-invasive breast lesions, performing immunohistochemistry in formalinfixed, paraffin-embedded core needle biopsy (CNB) samples. Materials and methods Tissue samples The expression of pro-Col11a1 was examined in 201 patients with radiological evidence of breast lesions. Samples were diagnosed as Infiltrating Ductal carcinoma (IDC, 87), Infiltrating Lobular Carcinoma (ILC, 14), other types of infiltrating carcinoma (4) like tubular or mucinous, Ductal Carcinoma in situ (DCIS, 19), Lobular Carcinoma in situ (LCIS, 6), Fibroadenoma (30), Columnar Hyperplasia (CH, 17) and other non-neoplastic lesions (24), including fibrosis, ductal calcifications, ductal dilatations and radial scars. A second population of 40 DCIS, which presents difficulties in the diagnosis of invasiveness, was selected to demonstrate the medical application of COL11A1 as a predictor marker for infiltration. Patient recruitment was conducted with written informed consent and under approval of the Clinical Research Ethics Committee of Cantabria (Mamacan-2012.049). COL11A1 immunohistochemical analysis Formalin fixed, paraffin embedded CNB samples were stained using proCol11a1 monoclonal antibody clone 1E8.33 (ONCOMATRYX, Bilbao, SPAIN). Roche-Ventana Ultra Benchmark automatized system (Roche, Oro Valley, Arizona, USA) was used to perform immunohistochemistry. Antigen retrieval was performed with Ultra CC2 buffer for 16 min at 98 ◦ C; antibody was diluted at 25 ␮g/ml and incubated during 30 min at RT. Optiview kit (RocheVentana) was used according to the manufacturer’s protocol. For the myoepithelial cell analysis, samples were boiled in target retrieval solution buffer (pH 9, 20 min; Dako, Glostrup, Denmark), subsequently cooled in distillate water and 1 × PBS. Next, the sections were incubated with primary monoclonal antibodies directed against Calponin (Calp clone, M3556, ready-to-use, Dako) and p63

Table 1 COL11A1 staining by lesion. COL11A1 staining

IDC ILC Other invasive carcinoma DCIS LCIS Adenoma CH Non-tumoral lesion

Positive

Negative

82 12 4 2 0 0 0 1

5 2 0 17 6 30 17 23

IDC: infiltrating ductal carcinomas, ILC: infiltrating lobular carcinoma, DCIS: ductal carcinoma in situ, LCIS: lobular carcinoma in situ, CH: columnar hyperplasia.

(DAK-p63 clone, IR662, ready-to-use, Dako). The antigens were visualized using biotinylated antibodies and streptavidin conjugated with horseradish peroxidase (EnVisionTM Mouse HRP, Dako). Diaminobenzidine (DAB, Dako) was used as the substrate. Staining was separately evaluated by two independent pathologists. COL11A1 was considered positive when at least one CAF presented clear immunostaining. A positive layer immunostains for myoepithelial markers was considered as indicative of no microinvasive foci. Statistical methods Fisher’s exact test was performed to analyze the staining difference between infiltrating and non-invasive lesions using SPSS 15 suite and GraphPad Prism (La Jolla, CA, USA). Results The mean age of patients was 56 ± 15 years, with a 25% percentile of 47 and a 75% percentile of 67 years. Fifty-three percent of the lesions were diagnosed in the left breast, while 47 percent were detected in the right breast (all data shown in supplementary Table 1). Immunolabeling of pro-COL11A1 was observed in CAF clusters. In particular, we found a higher concentration of staining cells in tumor expansion areas and disseminated among tumoral nests. We considered as positive any lesion presenting at least one stained CAF. Among infiltrating carcinomas, 94% showed positive staining (Fig. 1a and b), whereas the noninvasive lesions had only 3% immunostaining (p < 0.0001) (Fig. 1c and d). Table 1 presents the data obtained for each histologic subtype. Six samples were diagnosed as DCIS on CNB and presented staining for pro-COL11A1. Complementary surgical biopsy of this lesions presented invasiveness. The three false positive cases (two DCIS and one non-tumoral lesion) have not shown any recurrence to date (up to now) with a follow up of two years. Pro-COL11A1 showed a sensitivity of 94% and a specificity of 97% when used as a marker of infiltrating breast disease. Furthermore, expression of pro-COL11A1 in breast lesions had a likelihood ratio of 14. Positive predictive value for pro-COL11A1 was 0.97, while the negative value was 0.93. In order to determine the medical applicability of COL11A1, we decided to test a second series comparing COL11A1 expression with conventional markers of myoepithelial cells: calponin and p63. For these, 40 samples presenting difficulty on invasiveness diagnosis were selected. Although all of them were finally diagnosed as DCIS in CNB through the positive immunoexpression of myoepithelial markers, 21 of them presented microinfiltration (samples were selected with underdiagnosis for demonstrating the capacity of the new marker) in the subsequent surgical biopsy (Table 2, Fig. 2). The expression of COL11A1, however, allowed identification of microinvasion in the core needle biopsy. Nineteen out of 21 samples with

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Fig. 1. pro-COL11A1 expression in breast lesions. Immunostaining for pro-COL11A1 in: (a) Infiltrating Ductal Carcinoma, (b) Infiltrating Lobular Carcinoma, (c) Ductal Carcinoma in situ and (d) non-neoplastic lesion (columnar cell hyperplasia). Counterstained with Hematoxylin. Original images magnification 300×, inset images 600×

Table 2 Immunostaining for myoepithelial cells and Collagen XI ␣1. Calponin Positive Non invasive lesion Infiltrative lesion

p63 Negative

Discussion COL11A1

Positive

Negative

Positive

Negative 17

19

0

19

0

2

20

1

19

2

19

2

subsequent diagnosis of infiltration showed immunostaining for COL11A1, whereas only two of the 19 samples that were non invasive presented positive expression for COL11A1 (Table 2, Fig. 2). COL11A1 presents a significantly higher capability for predicting microinfiltration (in breast cancer CNB) than calponin or p63, with a sensitivity of 90% and a specificity of 89% (Table 3).

Table 3 Sensitivity and specificity of different invasion markers.

Sensitivity Specificity Positive predictive value Negative predictive Value

Calponin

p63

COL11A1

4.8% 100% 1 0.49

9.5% 100% 1 0.5

90% 89% 0.90 0.89

The results shown in this article reveal that the expression of pro-COL11A1 has a statistically significant association with invasiveness and malignancy in breast lesions. This marker presents a sensitivity and specificity higher than 90%, which makes it an excellent candidate for the differential diagnosis of breast-infiltrating lesions. The diagnosis of DCIS and other in situ lesions on CNB may present a challenge on the everyday-work of the pathologist. Several studies published [35–37] demonstrate a great underestimation in the diagnosis of infiltrative lesions. A meta-analysis published by Brennan et al. [38] assures that more than 25% of CNBdiagnosed DCIS presented infiltration (IDC) in the post-operative biopsy in a population of over 7000 cases. In order to assist the pathologist to afford (in delivering) a proper diagnosis, several differential markers between infiltrating and noninvasive lesions have been suggested. In a first attempt, loss of the basement membrane (BM) was proposed as an indicator of tumor invasiveness. However, immunostaining of the BM components, such as laminin and collagen IV, could be problematic since in situ lesions show variable BM loss, and invasive foci may remain at least partially surrounded by BM [39]. Furthermore, although there is a greater expression in non-invasive lesions, infiltrating tumors still express enough protein to be detected by immunohistochemical techniques [40,41].

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Fig. 2. Immunostaining comparative between in situ lesions diagnoses by Core Needle Biopsy. ICSB: Infiltrative carcinoma in subsequent surgical biopsy. Metastasis: In situ carcinoma in subsequent surgical biopsy. Lower case letters original magnification 300×. Capital letters original magnification 600×

The loss of the myoepithelial cells (MEC), present in the mammary gland, is a key event in the transition of in situ to infiltrating lesion. Therefore, several markers have been generated to demonstrate their presence [42]. S-100 protein was the first MEC marker, supposed not to be expressed in infiltrating breast tumors; however, some neoplastic cells have been shown to be positive for S-100 protein expression even in the absence of MEC [43,44]. Furthermore, it has been demonstrated that other MEC markers, such as high molecular weight cytokeratins 5, 6, 14 or 17, although they are very specific markers of invasiveness, present a low sensitivity, with a high false negative rate [45,46]. P63, although it does not have cross-reactivity with myofibroblasts, due to its nuclear label, may be lost due to the histologic section. Finally, other proteins such as heavy chain myosin for smooth muscle (HCMSM) or calponin have been described as good candidates for the differential diagnosis of infiltrating lesions. However, the rapid metabolism of calponin [47] and the cross-reactivity of HCMSM with myofibroblasts [48,49], reduce their sensitivity and specificity as markers of invasiveness. Therefore, new tools must be developed to enable the pathologist to provide an accurate diagnosis of every infiltrating lesion. At this point, pro-COL11A1 provides several advantages over the previously mentioned markers. The first and most important advantage of pro-COL11A1 is that it acts as a positive marker. Indeed, ECM and basement membranebased diagnosis are related to inactivation of protein expression in invasive lesions and, as a consequence, provide a negative result

in immunohistochemistry, while pro-COL11A1 is expressed only in infiltrating carcinomas. Moreover, infiltrating lesions are usually associated with an in situ component [39], which may generate difficulties in the interpretation of results when searching for loss of expression of a marker. Pro-COL11A1 is easier to analyze, since the presence of immunolabeling is directly associated with tumor invasiveness. Pro-COL11A1 is a marker expressed in fibroblasts dispersed among the tumor stroma, unlike MEC markers. This also represents a great advantage, since CNB samples are relatively small and limited, and therefore, basal membrane focal lesions might be under-recognized while only in situ lesions could be detected [38,50]. In contrast, almost all CNB samples present areas on which CAFs can be found. In this respect, our DCIS-underestimated series confirm that 90% of the DCIS on CNB that afterwards showed invasive foci in surgical proceeding were positive for COL11A1, warning of a possible infiltration, while neither p63 nor calponin showed a loss of signal. Even in the absence of infiltrative focus in CNB, the label for COL11A1 is able to predict invasiveness. The third advantage resides in its simplicity of use, based on a yes/no mode of interpretation. Other markers, such as calponin and myosin [42], have to be performed together for a correct diagnosis and are often subjected to changes in intensity of immunostaining. Pro-COL11A1 presents a sufficient sensitivity and specificity to discriminate non-invasive from infiltrating lesions with a dichotomous quantification -positive or negative-, considering positive a sample with the only presence of a single stained fibroblast.

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Several studies based on gene expression propose COL11A1 as a marker to discriminate infiltrating from non-invasive breast lesions [33,34]. However, a paper published by Halsted et al. [51], in contrast to our data, argues that there is COL11A1 expression in the normal breast and non-invasive breast lesions, whereas its expression is reduced when analyzing infiltrating tumors, even though the characterization of protein expression in these articles has been carried out in limited series of patients. Our work contributes with the use of a large CNB-based population as a closer approach, and secondly but most importantly, the use of a new highly specific antibody anti pro-COL11A1 [52]. Indeed, COL11A1 presents a high homology with COL5A1, not only structural but also functional [22]. Both collagens 5 and 11 regulate the diameter of the two major collagens type I and II (respectively [21]. Therefore, the use of an antibody against a specific region of COL11A1 avoids cross-reactivity with COL5A1, an thus generation of false positives. Moreover, the use of a monoclonal antibody further enhances specificity [52], and the use of the procollagen form provides an intracellular cytoplasmic staining pattern, much easier and specific to evaluate with respect to extracellular signal. In conclusion, this paper presents a series of evidence showing a strong correlation between the expression of pro-COL11A1 in cancer associated fibroblasts and invasiveness in breast lesions. We suggest that pro-COL11A1 antibody should be implemented as a complementary tool in the routine practice of the pathologist’s work for the analysis of breast lesions. Funding This study was supported by grants from the Government of Spain through the INNPACTO program, project Mamacan IPT 20111817-900000. Authors’ contribution All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Competing interests Both Saioa Domínguez-Hormaetxe and Laureano Simón claim to be employees of Oncomatryx. Rest of the authors declare that they have no competing interests. Acknowledgements We are grateful to Ms. Pilar García, Ms. Ainara Azueta and Ms. Irene González-Rodilla for their selfless help in the diagnosis of complicated cases. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.prp.2014.07.012. References [1] R. Siegel, D. Naishadham, A. Jemal, Cancer statistics, CA Cancer J. Clin. 62 (2012) 10–29. [2] C. DeSantis, R. Siegel, P. Bandi, A. Jemal, Breast cancer statistics, CA Cancer J. Clin. 61 (2011) 409–418. [3] R. Kalluri, R.A. Weinberg, The basics of epithelial-mesenchymal transition, J. Clin. Invest. 119 (2009) 1420–1428. [4] D.G. Tiezzi, S.V. Fernandez, J. Russo, Epithelial mesenchymal transition during the neoplastic transformation of human breast epithelial cells by estrogen, Int. J. Oncol. 31 (2007) 823–827.

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