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ORIGINAL ARTICLE  
Year : 2016  |  Volume : 59  |  Issue : 4  |  Page : 463-468
Evaluation of pathological parameters and morphometric data of desmoplastic lobular breast carcinoma


1 Institute of Pathology, Faculty of Medicine, University of Niš, Zorana Đinđića 81, 18000 Niš, Serbia
2 Faculty of Medicine, University of Niš, Zorana Đinđića 81, 18000 Niš, Serbia
3 Department of Physiology, Faculty of Medicine, University of Niš, Zorana Đinđića 81, 18000 Niš, Serbia
4 Department of Chemistry, Faculty of Science and Mathematics, University of Niš, Višegradska 33, 18000 Niš, Serbia

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Date of Web Publication10-Oct-2016
 

   Abstract 

Background: Invasive lobular breast cancer (ILC) is the second most frequent form of breast cancer. While cancer cells are regularly investigated, tumor stroma represents a highly unexplored field. Aims: The aim of this study is to perform a detailed investigation of clinical, immunohistochemical, and morphometric characteristics of desmoplastic (D) and nondesmoplastic (ND) ILC. Materials and Methods: This study included twenty cases of ILC that were divided into two groups designated as D and ND groups. Medical histories and diagnosis data were obtained from the archives of the Center of Pathology, Clinical center Niš (Serbia). Morphometric analysis of hematoxylin and eosin stained slides was performed using ImageJ software, and the obtained data were further statistical processed. Results: Statistical analyses of the data revealed that no significant differences between D and ND groups when patient age, estrogen receptor (ER), and progesterone receptor (PR) expressions and morphometrical parameters (such as the distance between groups of cancer cells and nucleocytoplasmic ratio) were compared. However, D and ND groups statistically, significantly differed in the occurrence of axillary lymph node metastasis, and when the ER and PR data were included, in certain nuclear parameters (cell/nucleus area, perimeter, Feret's diameter, and circularity). Conclusions: Desmoplastic stroma was observed more frequently in patients without axillary lymph node metastases, whereas the expression of ER and PR had no influence on its development. According to the measured morphometric parameters larger cells/nuclei belonged to ND group.

Keywords: Desmoplastic, lobular breast cancer, morphometry, nuclear area

How to cite this article:
Ilic IR, Stojanović NM, Randjelović PJ, Mihajlović MN, Radulović NS, Ilić RS. Evaluation of pathological parameters and morphometric data of desmoplastic lobular breast carcinoma. Indian J Pathol Microbiol 2016;59:463-8

How to cite this URL:
Ilic IR, Stojanović NM, Randjelović PJ, Mihajlović MN, Radulović NS, Ilić RS. Evaluation of pathological parameters and morphometric data of desmoplastic lobular breast carcinoma. Indian J Pathol Microbiol [serial online] 2016 [cited 2020 Oct 29];59:463-8. Available from: https://www.ijpmonline.org/text.asp?2016/59/4/463/191775



   Introduction Top


Breast cancer (BC) is the most frequent malignant disease of women in the modern world and with the incidence of almost one million new cases per year. Upper lateral quadrant and glandular tissue toward axillar region are the predilection sites for the development of BC. The most common type of BC is the ductal one (70% of all cases), whereas the remaining cases are diagnosed as lobular (15%) or other types of BC (<15%). The development and progression of tumor tissue are considered to be the consequence of the misbalance between apoptosis and proliferation of transformed cells. [1] This progression is also dependable on the interaction of transformed cells and the surrounding tissue. These interactions cause changes in the tumor stroma potentially leading to, among others, a desmoplastic reaction. [2]

Desmoplastic stromal phenotype, where most of the cells are fibroblasts, can be in a form of a dense fibrous scar, making up more than 50% of the entire tumor mass. A desmoplastic reaction is characterized by the presence of myofibroblasts in the stroma that synthesize and organize the extracellular matrix that is rich in fibronectins and collagen I. [3] The presence of desmoplastic (D) stroma is known to be associated with drug resistance, for example, adriamycin, of mammary carcinoma cells. [4]

Determination of hormone receptor expression (estrogen and progesterone) in lobular BC gives new diagnostic and prognostic data that can further be compared with morphometric parameters. Analyses of morphometric and immunohistochemical characteristics of lobular BC are scarce, especially in the domain of tumor stroma. Motivated by this, the aim of the current work was set to analyze and compare the pathological parameters and morphometric data of D and nondesmoplastic (ND) lobular BC.


   Materials and Methods Top


Study design

In this study, the pathological parameters and morphometric data from a total of twenty female patients with lobular BC, in the period from 2007 to 2009, were analyzed. The patients were divided into two groups of 10 patients, the first group was comprised patients that displayed a desmoplastic reaction of stroma (the study group), whereas the second group included patients without a desmoplastic reaction (the control group). Tissue samples of invasive lobular BC (ILC) were obtained by breast-conserving surgery or total mastectomy with an axillary dissection from the Clinical Center Niš and other clinical centers from southeastern Serbia. Samples were routinely processed, embedded in paraffin and archived together with their corresponding histopathological diagnosis and clinical documentation in the Center of Pathology of the Clinical Center Niš. From all tissue samples, a minimum of five sections were taken from the tumor, except when the tumor was <2 cm in size when the entire tumor mass was processed. When the desmoplastic stroma, predominantly connective tissue present, contributed to the tumor mass with more than 50% the tumors were considered to show a desmoplastic phenotype. [5] The data concerning patient age, marital status, working position, serum biochemical analysis, patient survival and the presence of metastases in axillary lymph nodes and other disease characteristics were obtained from the archive of the Center of Pathology of the Clinical Center Niš.

Immunohistochemical staining procedure

The characteristic areas of tumors (five paraffin blocks per case), from microscopically selected samples (regions), based on standard hematoxylin and eosin (H and E) staining, were immunohistochemically stained. Tissue from the paraffin molds was cut into 4 μm thick sections, placed on superfrost glass slides and stained immunohistochemically for the presence of estrogen receptors (ERs; Monoclonal Mouse Anti-Human ER-α; Clone 1D5; Code N1575, ready-to-use; DAKO, Glostrup, Denmark) and progesterone receptors (PRs; Monoclonal Mouse Anti-Human PR; Clone PgR 636; Code N1630, ready-to-use; DAKO, Glostrup, Denmark). After the sections were processed, according to the manufacturers directions, visualization was effectuated using diaminobenzidine and counterstained with Mayer's hematoxylin.

Scoring system

Scoring was done on at least 100 cancer cells per slide where medium to strong staining of nuclei was considered as positive. Cancer cells stained with ER or PR antibodies were considered positive for these markers when 1% of the cancer cells with strong nuclear staining were present or 10% of the cancer cells showed medium to weak nuclear staining (Allred scoring system).

Morphometric analysis

Images of microscopic slides, stained routinely with H and E, were captured using BH-2 microscope (OLYMPUS, Japan) equipped with a digital camera (OLYMPUS, E-450). Five images per slide were taken, under the magnification 400; the images were further analyzed using ImageJ software (imagej.nih.gov/ij/). The measured parameters included the distance between groups of cancer cells (μm; 10 shortest distances per captured image), and the area, perimeter (B), Feret's diameter and circularity (Cir) of the cells/nuclei. For the five images taken per slide, with minimum five slides per case, 15 cells/nuclei were measured. The analyzed cells had a clear cell membrane and did not overlap with the surrounding cells. The fields that were taken into consideration were on the periphery of tumor tissue where the boundary with normal breast tissue was visible.

Statistical analysis

The acquired data were processed using the descriptive statistical and comparative (Student's t-test and Fisher's test) analysis. The following statistical packages were used Jandel Sigma Stat 2.0 (SPSS Inc., Chicago, IL, USA) and GraphPad Prism version 5.03 for Windows (GraphPad Software, San Diego California USA, www.graphpad.com).


   Results Top


Out of a total of 20 patients included in this study, the case group consisted of lobular BCs with a pronounced desmoplastic reaction of the tumor stroma [Figure 1]a, whereas the control group was comprised patients with ND lobular BC [Figure 1]b. Furthermore, the mammographic images of BCs with or without a desmoplastic reaction are shown in [Figure 1]c and d, respectively. All patients were married, whereas their working positions were housewives, nurses, and professors. None of the standard biochemical parameters in blood/serum of these patients was altered during the BC diagnosis. Postoperatively, several patients had cancer-biomarker (CEA and CA 15-3) levels lower that the limiting values.
Figure 1: Histological and radiological presentation of desmoplastic (a and c) and nondesmoplastic (b and d) lobular breast cancers

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Out of 185 patients diagnosed with ILC, in the period from 2007 to 2009, only 10 (5.4%) had a desmoplastic stromal reaction. The data concerning average patient age from both groups, as well as the average age of patients with positive/negative ER/PR expressions are shown in [Table 1]. According to the Student's t-test, patients' age neither statistically, significantly, mutually differed between these two groups nor between the groups with different steroid receptor expression (P > 0.05; [Table 1]).
Table 1: Average age of patients with desmoplastic and nondesmoplastic lobular breast cancer

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Statistical analysis of the data revealed a significantly (Fisher's test, P = 0.0325) more frequent occurrence of axillary lymph node metastases in patients with desmoplastic lobular BC [Table 2]. Furthermore, other distant metastases or the presence of multicentricity were not noticed in the two study groups of patients with lobular BC.
Table 2: Relation between desmoplastic and nondesmoplastic lobular breast cancer and the presence of axillary lymph node metastasis

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Fisher's test showed no significant differences between the groups of patients with D and ND lobular BC with regard to the positivity of ER and PR expressions [Figure 2].
Figure 2: Fisher analysis of data dealing with the positivity of estrogen receptor and progesterone receptors in patients with desmoplastic and nondesmoplastic lobular breast cancer

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[Table 3] presents the measured distances between cancer cell groups, expressed in micrometer. No statistically significant differences were found between the measured distances in these two patient groups; however, the maximal distance was found to be greater in the case group (desmoplastic BC) compared to the control one [Table 3].
Table 3: Distances between cancer cell groups in micrometer (measured using ImageJ software)

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The data concerning the measured cancer cell and nuclear parameters, along with the corresponding P values, from both groups are shown in [Table 4]. The values of the cell and nucleus area, perimeter, and Feret's diameter were invariantly greater for the ND group when compared to the D group (P < 0.0001). In the case of nuclear circularity, statistically, significantly rounder cancer nuclei were found in the D BC group [Table 4]. Nucleocytoplasmic ratio did not differ between the groups [Table 4].
Table 4: Comparison of data regarding the cell/nuclear morphometric parameters of cancer cells in desmoplastic and nondesmoplastic breast cancer groups

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Correlations between the ILCs nuclear grade (NG)/hormone receptor positivity and mean nuclear area/perimeter are shown in [Table 5]. Detailed statistical analysis of the previously mentioned data is shown in [Table 6]. The analyzed nuclear parameters statistically, significantly differed between D and ND groups only when the ER and PR data were taken into consideration [Table 5] and [Table 6].
Table 5: Comparison of nuclear grade/hormone receptor positivity and mean values for the nucleus area (μm2) and perimeter (μm)


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Table 6: Statistical comparison (Student's t- test) of data concerning the nuclear grade/hormone receptor positivity and mean values for the nucleus area (μm2) and perimeter (μm)

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   Discussion Top


Investigations of the desmoplastic reaction of stroma could contribute to the better understanding of molecular biology of certain BC types such as the invasive ductal BC (IDC). Although this type is characterized by pronounced stromal reaction, similar reactions could be seen in other types, i.e., ILC. [6]

Until now, no reports were published on the correlation between patient age and either the presence of a desmoplastic reaction or the expressions of hormone receptors (ER and PR). Our study did not show any significant differences in age-related data between the two studied groups [Table 1]; however, the obtained results, most certainly, contribute to the overall findings in this field.

Desmoplasia is characterized by the presence of collagen fibers, myofibroblasts, and mesenchymal cells mixed with smooth muscle cells and fibroblasts. [1] Furthermore, versican and other proteoglycan matrices (tenascins, fibronectins, and integrins) have been studied in breast pathology and are proved to be present in the desmoplastic stroma in the vicinity of invasive and preinvasive lesions of breast tissue. [7],[8] Although the exact mechanism of induction of the desmoplastic reaction is still unknown, [9],[10] there are newer studies that suggest that vascular endothelial growth factor (VEGF) expression is connected with this type of stromal reaction. [11] This is pronounced in the case of +ER cancers where the VEGF over-expression enhances tumor-stroma interaction and allows stroma adaptation and tumor metastasis even with the tamoxifen hormonal therapy applied. [11] Furthermore, the worst disease prognosis is associated with highly proliferative fibroblasts and stroma-rich in fibronectins and collagen I. [12] The expression of thrombospondin-1, responsible for the promotion of invasion and metastasis of some cancers, was found to be significantly more positive in patients with desmoplastic ductal BC. Besides this, its potential role in the promotion of lymph node metastasis in ductal cancers emphases its role as a predictive marker for metastasis. [13] This increased metastasis frequency of occurrence related to the ductal BC does not appear to hold for the lobular BC since we demonstrated a statistically more frequent metastasis incidence for the control group when compared to the case group [Table 2]. Newer publications also point to the significant correlation between myofibroblastic stromal reaction and the presence of lymph node metastases. [14] The presence of desmoplastic stromal reaction seems to be preventing the lymph node metastases, indicating that it may be a useful predictive parameter due to that fact that neither one of our patients had metastases in axillary lymph nodes.

In in vitro studies of BC cells cultured in the presence of estrogen are deprived from stromal tissue. On the other hand, the absence of estrogen in various BC cultures lead to a poor development of cultures and in one case caused a significant desmoplastic reaction. [15] Although the obtained data suggest that the ER expression is a characteristic of desmoplastic BC (80% of cases; [Figure 2]), the increased lymph node metastases were not observed as expected. [11]

The majority of knowledge about cell and nuclear morphology is in a descriptive form, and only a modest amount of numerical data exists. [16] Several publications, dealing with the differences between IDC and ILC, showed a few morphometric features helpful in the distinguishing between ILC and IDC. [16],[17],[18] Among these parameters, the mean nuclear area is the most reliable in distinguishing benign versus malignant lesions. [18] The cellular and nuclear morphometric parameters of ILC were proven to differ from IDC parameters. [16] Mean values for the measured cell and nuclear parameters significantly differed for the two groups of cancer cells (in most cases P < 0.0001; [Table 4]). The data point to the fact that the cells/nuclei in the desmoplastic BC group were smaller than those from the control group. Furthermore, a statistically, significantly greater nuclear area of the ND-BC cells can be the consequence of a higher proliferative index of BC. The study that dealt with the comparison of mean nuclear area and perimeter between different NG IDC showed the existence of statistically significant differences between the area and perimeter values in the three grade groups. [19] This was not the case in our study, as the analysis failed to establish a difference between D and ND nuclear morphometric values [Table 6]. A greater number of studied cases (there was a high SD in the herein studied groups) might resolve this discrepancy.

No statistically significant differences were found between the morphometric values for different ER and PR expressions in the desmoplastic BC group [Table 6]. However, there was a statistical difference noted between the measured parameters in D and ND groups when the expression of ER and PR was taken into consideration [Table 6]. It seems that the nuclei in ER–/PR–D group were larger than in the ER+/PR+ within the same group (although no statistical significance was found for this difference). Furthermore, there was no statistical difference between the mean area values in the ER–/PR– D group and the group ND-BC with ER+/PR– or ER+/PR+, that had larger nuclear areas than D BC with ER+/PR+ [Table 6]. These data are not easily interpreted when having in mind that the ER and PR are located in the cell nucleus and their mode of action includes a vast number of hormonal effects that reflect on protein, RNA and DNA increase which have an impact on nucleus size. [20] The possible explanation of the obtained results may lay in the fact that in the advanced stage of BC there are numerous populations of cancer cells with different clones and aggressiveness potential. Different clones can have different levels of p53 expression, DNA ploidy, levels of proliferation, and nuclear morphology. [21] Trying to avoid all this, cells from the invasive front, where the cell morphology tends to be more unique and probably all the important receptors are expressed, have been analyzed. [22],[23]


   Conclusion Top


It was found that the desmoplastic stroma occurred more frequently in patients without axillary lymph node metastases and that there was no correlation between ER and PR expressions and the occurrence of the desmoplastic stroma. Interestingly, we did not find statistically significantly greater distances between tumor cell groups in patients with desmoplastic stroma when compared to those from the control group. However, the maximal distance between groups of cancer cells was much greater in desmoplastic than in those with ND stroma. Some of the values for the measured morphometric cell and nucleus parameters were shown to differ between D and ND-BC, indicating statistically larger cells/nuclei in the ND group.

Acknowledgment

This work was funded by the Ministry of Education, Science and Technological Development of Serbia (Project 172061 and 175092).

Financial support and sponsorship

This work was funded by the Ministry of Education, Science and Technological Development of Serbia (Project 172061 and 175092).

Conflicts of interest

There are no conflicts of interest.

 
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Correspondence Address:
Ivan R Ilic
Institute of Pathology, Faculty of Medicine, University of Niš, Zorana Đinđića 81, 18000 Niš
Serbia
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0377-4929.191775

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