Background
Primary signet ring cell carcinoma of the breast (SRCC) was first described by Saphir as a mucinous carcinoma [
1], and to date, few cases of primary signet ring cell carcinoma of the breast have been reported. The diagnostic criteria for SRCC have not been strictly defined either, since marked signet ring cell differentiation can occur in invasive lobular carcinoma, nonspecialized invasive carcinoma, and other specialized types; therefore, the WHO classification of breast tumors does not classify SRCC as a distinct disease entity, and the WHO changed the name of SRCC to carcinoma with signet ring cell differentiation. For convenience, we still use SRCC in the following description.
However, tumors with signet ring-like cells can occur in many organs of the body, such as the digestive tract, especially the common gastric signet ring cell carcinoma, the salivary glands, lungs, ovaries, etc. [
2‐
4]. Therefore, we need more specific markers for each tumor site to distinguish whether cancer with signet ring cell differentiation has metastasized to the breast. Aberrant expression of the membrane-bound form of mucin, MUC1, and the secreted form of mucin, MUC2, may be associated with cancer growth, differentiation, transformation, and invasion [
5]. However, the role of mucin in primary breast cancer with signet ring-like cell differentiation remains to be further investigated.
In this article, we analyzed breast cancer cases with signet ring cell differentiation. The histological morphology of each case was carefully described. We evaluated the staining pattern of several common immunohistochemical markers, including markers commonly used in breast cancer including Estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER-2), gross cystic disease fluid protein15 (GCDFP-15), and GATA binding protein 3.
(GATA-3); epithelial and myoepithelial expression patterns including CK7, CK20, and CK5/6; and especially the relative specific markers of the digestive system, including CDX2, cyclooxygenase 2(COX-2), Specially rich in AT sequence binding proteins (SATB-2), and Villin. Lung adenocarcinoma-related markers were evaluated including Thyroid transcription factor-1 (TTF-1) and Napsin A, and ovarian cancer-related markers including pair box gene 8 (Pax-8) and Wilm’s tumor gene 1(WT-1). The expression of neuroendocrine factors was also described. Differences in morphological and biological behaviors among different cases of breast cancer with signet ring cell differentiation and differences in immunohistochemical expression between breast cancer with signet ring cell differentiation and signet ring cell carcinomas derived from other sites have been discovered and identified. At the same time, the expression patterns of mucins, including MUC1 and MUC2, were found to be different from those of the surrounding concomitant cancer tissues. By Alcian Blue (pH 2.5)–Periodic Acid–Schiff (AB-PAS) staining, it was found that the characteristics of the mucus in the signet ring cells in the same case were also different.
Discussion
In clinical practice, signet ring cell carcinoma mostly occurs in the gastrointestinal tract. Primary breast signet ring cell carcinoma (SRCC) is very rare. In 2003, the World Health Organization (WHO) classified breast cancer and classified SRCC as the latter “other types” of mucinous carcinoma (special type cancer) and other tumors with rich mucus. In 2012, the WHO changed the name of SRCC to carcinoma with signet ring cell differentiation in the new edition of the “WHO Classification of Breast Tumors” and no longer as an independent type [
6]. Our study reported that breast cancer with signet ring cell differentiation can accompany ductal carcinoma, lobular carcinoma, and mucinous carcinoma, but the proportion of signet ring cells varies. Then, the question comes again. There is no unified conclusion about the proportion of signet ring cells that can be diagnosed as cancer with signet ring cell differentiation. Some scholars believe that to make a diagnosis of cancer with signet ring cell differentiation, signet ring-like cells must account for ≥ 10%, and the percentage of signet ring cells should be indicated in the report and that this may be related to poor prognosis. Another study compared three different cutoff values of 20%, 30%, and 40% and believed that 30% could be better related to clinical parameters [
8]. The five cases in this paper have been strictly selected. Although the proportion of signet ring cells in these 5 cases ranged from 10 to 80%, the morphology of the cells fully conformed to the definition of the WHO 2012 version and was confirmed to contain intracellular mucus by AB-PAS staining.
Most primary breast cancers with signet ring cell differentiation have a breast mass as the primary symptom. Compared with other common types of invasive breast cancer, cancer with signet ring cell differentiation is more aggressive, has a higher rate of lymph node metastasis, and has a poor prognosis [
9,
10]. Our cases showed that all 5 patients were admitted with breast lumps of different sizes, and two patients had obvious clinical symptoms with redness, pain, and nipple discharge. In two of these cases, vascular invasion and metastases to lymph nodes were found. Although we strictly selected all the cases, the nuclei of the signet ring cells still had various characteristics. Some cases had the same nuclear morphology, and some cases had large differences in the nuclei, with deeper staining, more intense chromatin staining, and different nuclear grades; there were also differences in the arrangement. In some cases, the adhesion between the cells was very poor, and the cells are scattered, while in some cases, the cells were arranged in sheets, clusters, or cords; similarly, the interstitium between the cells was also different, and some were rich in external mucus, and some were divided by fibrous stroma. Whether these differences are closely related to the prognosis of the tumors or due to the lack of our case data, further study of case accumulation is needed.
Molecular subtypes of breast cancer have important implications for the individualized treatment of patients. Our report shows that primary breast cancers with signet ring cell differentiation almost always express ER, PR expression is variable, and HER2 is negative in all the cases; therefore, primary breast cancers with signet ring cell differentiation are either luminal A or luminal B, and the positive index of Ki-67 also reflects this. However, because one of the cases of luminal type A had a large tumor (9.5 cm in diameter), vascular invasion, and lymph node metastasis occurred. The staging was late, and death occurred after 8 months of follow-up. This may also indicate that primary breast cancer with signet ring cell differentiation, even if it is luminal A, is still more aggressive and has a worse prognosis.
It is also crucial in distinguishing signet ring cell carcinoma from breast tissue primary from other sites. Regardless of where SRCC originates, it often metastasizes to regional lymph nodes, the peritoneal surface, ovaries, and lungs [
11]. Therefore, this paper discusses the identification of primary breast cancer with signet ring cell differentiation from tumors derived from the digestive tract, lung, ovary, and other organs. First, in terms of epithelial and myoepithelial expression, CK20 is usually expressed in gastrointestinal adenocarcinoma and ovarian mucinous tumors, while SRCCs are all negative, which has identification significance. Since CK7 is expressed in most SRCCs and negatively expressed in gastrointestinal adenocarcinomas, it also has identification significance, and due to the different cell morphology of CK7 in signet ring cells, the abundant mucus in the cells may weaken its expression, but further research is needed. The myoepithelial markers CK5/6 were of little value in identifying the source. Primary breast tumors have more specific markers, such as GATA3 and GCDFP-15, and the former has a higher positive rate and is more meaningful in differentiating tumors from other sites, which is consistent with the results of previous studies [
12,
13]. However, GCDFP-15 is not completely useless. Although a small number of lung adenocarcinomas may also express it, it is still meaningful to differentiate it from some tumors, such as those of the gastrointestinal tract and ovary, when it is positively expressed in breast cancer. Our results show that the specific markers of gastrointestinal tumors, including CDX2 and SATB2, were consistently negative in SRCC, while Villin or COX2 had positive expression levels in one case, so this indicator needs to be carefully considered in the differential diagnosis. In the differentiation of tumors from the ovary, PAX8 is consistently negative in SRCC, while WT-1 is partially positive, so the latter is of little significance and cannot be used alone. The related markers of lung adenocarcinoma, including TTF1 and Napsin A, were both negative in SRCC, indicating that these two indicators are of great significance in the differential diagnosis. In conclusion, in differential diagnosis, we need to pay attention to particularly useful markers, such as ER, CK20, GATA3, CDX2, SATB2, PAX8, TTF1, and Napsin A, as well as to markers whose expression is not constant, and special care should be taken when these markers are expressed abnormally.
Neuroendocrine differentiation is more likely (up to 30%) in nonspecialized invasive breast cancers or other specialized types, especially mucinous carcinomas. Therefore, this paper also observed the expression of neuroendocrine markers such as Syn, CgA, and CD56 and found that one case expressed three neuroendocrine markers. This indicates that SRCC can also be associated with neuroendocrine differentiation. In general, breast cancer with neuroendocrine features has a worse prognosis than other types of breast cancer because neuroendocrine differentiation itself is an independent poor prognostic factor [
14]. Therefore, SRCC with neuroendocrine differentiation is also a problem that requires attention.
Mucins are divided into three groups according to their physiological characteristics: secreted, membrane-bound, and soluble mucins. The most typical representative membrane-bound mucin is MUC1. Ohashi et al. [
8] detected the expression of various MUC proteins in breast cancer with signet ring cell differentiation and found that MUC1 expression was divided into two modes: luminal margin plus cytoplasmic positive (LC) and cytoplasmic staining with cell membrane enhanced staining (MC), where CM patterns are often associated with poor clinicopathological factors. Our study showed that MUC1 was positively expressed in all the cases, but the expression pattern was different among the cases. Some cases had weakened expression intensity, some cases had a strong perimembranous expression, and the expression level was significantly higher than that of other types of invasive cancers accompanying the surroundings. These differences are likely to be related to the clinicopathology of tumors. However, due to the small sample size included in this study, effective statistical analysis cannot be performed, and further research is needed to increase the sample size in the future. As a representative secreted mucin, MUC2 is mainly expressed in colorectal goblet cells and colon and rectal cancer cells. Walsh et al. reported that MUC2-positive breast cancer patients had significantly shorter survival than patients with MUC2-nonexpressing tumors (49 months vs. 75 months) [
15]. Astashchanka Anna et al. reported that MUC2 plays an important role in mediating breast cancer cell proliferation, apoptosis, and metastasis. MUC2 may be important to guide treatment and predict outcomes in breast cancer patients [
16]. Our experimental results showed that MUC2 was strongly expressed in all the cases and was significantly higher than that in the peripheral invasive ductal carcinoma, lobular carcinoma, or mucinous carcinoma. Whether this is associated with a worse prognosis in breast cancers with signet ring cell differentiation remains to be investigated.
In addition, the results of AB (pH 2.5)-PAS staining in all the cases showed that breast signet ring cell carcinoma had greater heterogeneity, which not only manifested in the mixed existence of multiple histological morphologies in the same tumor tissue but also showed differences in the properties and amount of intracellular mucus in similar tumor tissues in the different cases or different mucus lakes in the same tumor tissue. We can see neutral mucus, acidic mucus, mixed mucus, and transitional morphologies in all the cases. Different signet ring cell carcinomas are either predominantly neutral mucus (red cells), acidic mucus (blue cells), or both (purple cells), but neutral mucus is generally the most common. The main signet ring cells are more abundant, and the extracellular mucus is more acidic. Yameshina [
17] confirmed that cancer cells have diverse shapes and different mucus reactions, indicating that cancer cells are in different stages. We believe that signet ring cell carcinoma has multiple cell subsets and retains the potential for multidirectional differentiation. The difference in mucus expression and traits of cancer cells may be due to the differentiation and development of stem cells at different stages or different cloned tumor cells. Thus, during tumor evolution, cancer cells express different mucinous traits. In addition, it is speculated that the influence of precancer environmental factors or stress also plays an important role in the heterogeneity of cellular mucus, which is also required for tumor cell phenotypes to change to suit their survival [
18].
In conclusion, although breast cancer with signet ring cell differentiation is no longer regarded as an independent type, these tumors still have their own characteristics, and their histological morphology is still different even if they are strictly defined. The molecular type is mainly luminal A or type B, and their expression of some immune markers is inconsistent with that of the surrounding ductal, lobular, and mucinous carcinomas. We need to be especially cautious when aberrant expression of nonconstant markers is used in differential diagnosis. The expression pattern of mucin markers may be related to the clinicopathological characteristics of tumors, such as tumor prognosis, and the characteristics of mucin in these tumor cells may also be closely related to the occurrence and evolution of tumors.
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