Triple-negative breast cancer: Histological subtypes and immunohistochemical and clinicopathological features

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1 Triple-negative breast cancer: Histological subtypes and immunohistochemical and clinicopathological features Yuko Ishikawa, 1,2 Jun Horiguchi, 2 Hiroyuki Toya, 1,2 Hiroki Nakajima, 1 Mitsuhiro Hayashi, 3 Nobumi Tagaya, 4 Izumi Takeyoshi 2 and Tetsunari Oyama 1,5 Departments of 1 Diagnostic Pathology, 2 Thoracic and Visceral Organ Surgery, Gunma University Graduate School of Medicine, Gunma; 3 Breast Oncology, Tokyo Medical University Hachioji Medical Center, Tokyo; 4 Second Department of Surgery, Dokkyo Medical University, Tochigi, Japan (Received August 10, 2010 Revised December 2, 2010 Accepted December 12, 2010 Accepted manuscript online January 8, 2011) To reveal heterogeneous properties of triple-negative (TN) breast cancers (estrogen receptor negative, progesterone receptor negative and HER2 negative) and to clarify whether the developmental pathways to TN breast cancer are single or multiple, we conducted clinicopathological and immunohistochemical studies on TN breast cancers, with special reference to comparison of the invasive component (iic) and the ductal component (dcic) of invasive TN breast cancer and pure TN ductal carcinoma in situ (TNDCIS). Tumor tissues were obtained from 97 patients with TN invasive carcinoma and 10 patients with TNDCIS. Two histological subclassifications, atypical medullary carcinoma (type A, n = 16) and carcinoma with a central acellular zone (type B, n = 11), were distinguished from conventional ductal carcinoma. Other invasive ductal carcinomas were classified as type C (n = 64) and special types were classified as type D (n = 5). The follow-up period for the 96 patients ranged from 5 to months (mean, 47.6 months). Out of 97 cases, dcic was present in 29 (30%) cases and type A and B had significantly few ductal components, 0% and 18%, respectively. There were only six (6%) cases with non-tn cells in dcic and TN cells in iic and five of them were type C. In 13 (13%) cases, epidermal growth factor receptor (EGFR) expression existed only in iic. Therefore, most of the TN carcinoma develops originally and rapidly invades at the early stage, especially in types A and B. The relapse rate of type B was the highest (36.4%) and the overall survival of patients with type B was the shortest (P = 0.02), which indicates that the prognosis of type B is significantly worse than the other types. (Cancer Sci 2011; 102: ) Breast cancer is a heterogeneous disease that includes various biological entities, which are separated on the basis of specific morphological and immunohistochemical features as well as clinical behavior. For many years, invasive breast cancer was classified according to its histological features and expression of hormone receptors (HR), that is, the estrogen receptor (ER) and the progesterone receptor (PR). More recently, characterization of HER2 status has become important for classifying invasive breast cancer with regard to trastuzumab treatment. Tumors that are negative for ER, PR and HER2 (triple negative [TN]) are reported to account for 12 25% of invasive breast cancer, and initially some of this group has attracted attention because of aggressive behavior and lack of response to currently available systemic therapy. (1 9) Recently, genome-wide microarray analysis has been used to classify invasive breast cancer at the transcriptional level into five main groups, which are luminal A, luminal B, normal breast-like, HER2 and basal-like. (10,11) The basal-like group includes many tumors without expression of HR and HER2, and some parts of this group show aggressive behavior and a poor prognosis. (1,3 5,8 11) Triple-negative breast cancer overlaps the profile of basal-like cancers. However, TN cancer and basal-like cancer are not exactly the same, because approximately 20 30% of TN tumors are negative for both EGFR and basal cytokeratins or are non-basal according to their global gene expression profile. (1,3,5,10) Therefore, TN breast cancer is another heterogeneous group and CK5 6, CK14 and EGFR negative tumors are reported to be biologically different from TN basal-like tumors. Regarding the histological features of TN tumors, the majority are non-special ductal tumors (invasive ductal carcinoma) with a high level of nuclear atypia, which is grade III, while other reported types are medullary carcinomas, metaplastic carcinoma and adenoid cystic carcinoma. (1) Medullary carcinomas, including typical and atypical medullary carcinomas, have been reported to show BRCA1 germ-line mutation and these tumors are characterized by a high level of basal myoepithelial differentiation. (12,13) Another important histological feature of highgrade invasive ductal carcinoma is a large central acellular zone (CAZ), which was reported by Tsuda et al. (14 16) These tumors often differentiate toward a basal myoepithelial type. However, few studies have analyzed the pathological features of TN breast cancer, especially its detailed pathological subclassification. In this study, immunohistochemical and clinicopathological assessment of TN breast cancer was carried out to define its histopathological features. Furthermore, to reveal if histogenetic pathways to TN invasive breast cancers are single or multiple, we compared the immunophenotype between the ductal carcinoma in situ (DCIS) component (dcic) and the invasive component (iic) of TN invasive breast cancers. Materials and Methods Patients and tumor tissues. Out of 1606 patients with invasive breast cancer and 80 patients with DCIS who underwent surgery for breast cancer at Gunma University Hospital (Maebashi, Gunma) or Dokkyo University Hospital (Mibu, Tochigi) from 1996 to 2007, tumors with ER()), PgR()) and HER2 score 0, 1or 2 and HER2 CEP17 < 1.8 by FISH were selected by the pathological reports and the tumor tissues with enough material were immunohistochemically examined again. Patients with neoadjuvant chemotherapy and or distant metastases at initial diagnosis were excluded. Finally, tumor tissues were obtained from 97 patients with TN invasive carcinoma (IC) and 10 patients with TNDCIS. The clinicopathological features of the patients are shown in Table 1. The group with IC ranged from 27 to 89 years old, with a mean age of 56 years, while the patients with DCIS ranged from 34 to 75 years old and had a mean age of 58.1 years. There was no significant difference of 5 To whom correspondence should be addressed. oyama@med.gunma-u.ac.jp Cancer Sci March 2011 vol. 102 no doi: /j x

2 Table 1. Clinical and pathological characteristics Invasive carcinoma N % Patients (n) 97 Age (years) Mean 56 Range Nuclear grade Low Intermediate High pt stage Histopathological feature Invasive ductal carcinoma Apocrine carcinoma Medullary carcinoma Spindle cell carcinoma Small cell carcinoma Lymph node metastasis Absent Present Unknown Intraductal component Absent Present pstage I II III Unknown Ductal carcinoma in situ Patients (n) 10 Age (years) Mean 58.1 Range Nuclear grade Low 1 10 Intermediate 5 50 High 4 40 age between the two groups. In the IC group, the pt stage was pt1 in 51 patients (52.6%), pt2 in 42 (43.3%) and pt3 in four (4.1%). With respect to nuclear grade, the 97 IC tumors were classified as low grade in two patients (2.1%), intermediate grade in six patients (6.2%) and high grade in 89 patients (91.8%). The original histopathological classification of the 97 IC tumors was invasive ductal carcinoma (IDC) in 92 patients (94.8%) and special type in five patients (5.2%). In the IC tumors, positive lymph node metastasis was in 33 patients (34.0%), negative lymph metastasis was in 61 patients (62.9%) and unknown was in three patients (3.1%). Immunohistochemistry and evaluation of immunostaining. Surgically resected specimens were fixed in 10% formalin and embedded in paraffin for routine pathological examination. We cut 4-lm thick sections from a paraffin block containing representative tumor tissue. Immunostaining for ER, PgR, p53 and MIB-1 was performed with the Ventana XT System (Ventana Japan, Tokyo, Japan) using the antibodies listed in Table 2. Immunostaining for cytokeratin 5 6 and cytokeratin 14 was performed by the ABC method using the antibodies in Table 2 after antigen retrieval (heating in a microwave oven at 95 C for 10 min). Immunostaining for EGFR was performed with a DAKO EGFR pharmdx kit (Dako North America Inc, Carpinteria, CA, USA) according to the manufacturer s instructions. Tumors with an Allred score 3 and nuclear staining of more than 10% of the tumor cells were classified as ER(+) or PgR(+). HER2 was defined as positive when the tumor had a score of 3+ according to the standard immunostaining scoring system or for tumors with a score of 2+ and HER2 CEP17 > 1.8 by FISH. EGFR was defined as positive when the score was 2+ or 3+, using the same criteria as for HER2. P53 was defined as positive when more than 10% of tumor cells were positive for nuclear staining, while CK5 6 and CK14 were defined as positive when more than 5% of cells were stained in the cytoplasm. (17 19) The MIB-1 index was calculated as the nuclear staining rate for more than 500 cells of IC and more than 100 cells of DCIS. Histological subclassification of breast cancer. In this study, the histological classification was based on the General Rules for Clinical and Pathological Recording of Breast Cancer (16th edition) of the Japanese breast cancer society. (20) However, two additional histological subclassifications were added for invasive carcinoma, because we sometimes encounter TN invasive carcinoma (TNIC) with characteristic histological features and such tumors have also been reported by several pathologists. (12,21 24) One of the new categories was typical and atypical medullary carcinoma (classified as type A) (Fig. 1), as defined by Ridolfi et al. (25) The criteria of medullary carcinoma are: (i) syncytial architecture; (ii) no glandular or tubular structures; (iii) diffuse lymphoplasmacytic infiltration; (iv) round carcinoma cells with vesicular nuclei and marked pleomorohism, mitosis; and (v) complete histological circumscription, as described in the 2003 WHO classification of Tumors of the Breast and Female Genital Organs. (26) Atypical medullary carcinoma is defined as tumors showing the association of a predominantly syncytial architecture with 2 3 of the other criteria. Atypical medullary features are common in BRCA1-associated carcinoma and these tumors frequently show a basal-like phenotype. (12) The other new category was carcinoma with CAZ Table 2. Details of the primary antibodies used in the present study Antigen Clone Dilution Source Positive score EGFR Monoclonal, 2-18C9 1:1 DAKO Cytomation, Glostrup, Denmark >10% ER Monoclonal, 6F11 1:1 Ventana Medical Systems, Inc., Tucson, AZ, USA >10% PgR Monoclonal, 16 1:1 Ventana Medical Systems, Inc., Tucson, AZ, USA >10% HER2 Monoclonal, CB11 1:1 Ventana Medical Systems, Inc., Tucson, AZ, USA >10% CK5 6 Monoclonal, D5 16 B4 1:100 DAKO Cytomation, Glostrup, Denmark >5% CK14 Monoclonal, LL002 1:20 NOVO castra Laboratories, Newcastle upon Tyne, UK >5% p53 Monoclonal, CM1 1:2000 NOVO castra Laboratories, Newcastle upon Tyne, UK >10% Ki-67 MIB-1 1:100 DAKO Cytomation, Glostrup, Denmark Positive rate EGFR, epidermal growth factor receptor; ER, estrogen receptor; PgR, progesterone receptor. Ishikawa et al. Cancer Sci March 2011 vol. 102 no

3 (A) (A) Fig. 1. Atypical medullary carcinoma (type A). (A) Low-power view (HE stain): broad anastomosing bands of pleomorphic tumor cells growing in a compact syncytial fashion. High-power view (HE stain): relatively narrow cords of syncytial cells with a high nuclear grade. Fig. 2. Tumor with a central acellular zone (CAZ; type B). (A) Lowpower view (HE stain): tumor cells have undergone infarction at the center, collagen can be seen. CAZ covered 30% of the entire tumor area. High-power view (HE stain): the border of tumor cells and a central acellular zone. (classified as type B) (Fig. 2), according to the classification of Tsuda et al. (15) This type of IDC is consisted to have undergone infarction at the center, and deposits of hyaline material and collagen occupy more than one-third of the cut surface of the tumor with sparse myofibroblasts, accompanied by a myxoid matrix at the periphery. This carcinoma shows no features of spindle cell carcinomas, osseous or cartilaginous metaplasia, or matrixproducing carcinomas. Recently, the presence of a CAZ has been noted in basal-like-type breast cancer. (1,16) Conventional invasive ductal carcinomas other than types A and B, including solid-tubular, papillotubular and scirrhous carcinomas, were classified as type C (conventional IDC) (Fig. 3A), while apocrine and metaplastic carcinoma were classified as type D (special types) (Fig. 3B). Statistical analysis. Statistical analysis was carried out with the Kruskal Wallis test using SPSS 11.0 software (SPSS Japan, Tokyo, Japan). Survival curves were drawn using the Kaplan Meier method and differences between the curves were calculated with the log rank test. Cox s proportional hazard method was used for univariate analysis to assess the risk of metastasis to each organ or cancer death. These analyses were performed using Statview 4.5J for Windows (SAS Institute Inc, Cary, NC, USA). Differences of P < 0.05 were considered to be significant. Results Immunohistochemistry of invasive carcinoma for multiple markers. EGFR expression was significantly correlated with CK5 6 expression (P = 0.04), and CK5 6 was correlated with CK14 (P < 0.001). Immunohistochemical features of TNDCIS, the ductal component and invasive component of invasive carcinoma (dcic and iic). We compared the immunohistochemical findings among DCIS, the ductal component of IC (dcic) and the invasive component of IC (iic) (Table 3). This analysis showed a significant difference in EGFR expression (P = 0.005) among the three groups. The EGFR-positive rate was 50% for DCIS, 24% for dcic and 59% for iic. Positivity of iic for EGFR was significantly higher than that of dcic (P = 0.001). The CK14-positive rate was 30% for DCIS, 69% for dcic and 64% for iic, and the positivity of DCIS was significantly lower than that of dcic and iic (P = 0.046). P53 expression was 49.5% in iic, 48.3% in dcic and 30% in TNDCIS and there was no statistical difference. There was a trend for MIB-1 positivity to increase from DCIS to dcic and then to iic. 658 doi: /j x

4 (A) Fig. 4. Clinical and pathological characteristics among A to D histopathological subtypes of triple negative carcinoma of the breast. Fig. 3. Conventional and special types of cancer (types C and D). (A) Invasive ductal carcinoma (type C; low-power view, HE stain): papillotubular carcinoma. Apocrine carcinoma (type D), low-power view, HE stain): tumor cells with apocrine differentiation. Table 3. Correlation of immunohistochemical markers between invasive and ductal components of invasive carcinoma (IC) and DCIS *P-value <0.05; **P-value < Nuclear staining rate (%). DCIS, TN ductal carcinoma in situ; dcic, ductal components of TN invasive breast cancer; iic, invasive components of TN invasive breast cancer; TN, triple negative. Existence of ductal components (dcic) and immunohistochemical findings of the four histopathological subtypes (A to D). Out of 97 cases, dcic was present in 29 (30%) cases. There was a significant difference with respect to the presence of a ductal component, because type A had no ductal component (0 16) and only 18.2% (2 11) of type B had a ductal component, while it was found in 35.9% (23 64) and 60% (3 5) of types C and D, respectively (P = for A, B vs C, D). Types A and B had a significantly higher MIB-1 index (58% and 62.3%, respectively) compared with types C and D (P = 0.002) (Fig. 4). In this study, inconsistent patterns of ER PgR HER2 and EGFR expression between dcic and iic were observed in 15 out of 29 subjects. Estrogen receptor expression was not consistent between dcic and iic in five patients, with dcic being positive and iic being negative in all of them (Fig. 5). Four out of the five cases were type C and one case was type D. One patient had a positive ductal component and a negative invasive component for PR and HER2 expression (both were type C). After all, there were only six (6%) cases with non-tn cells in dcic and TN cells in iic and five of them were type C. EGFR expression was different in 13 patients, with dcic being negative and iic being positive in all of them (Table 4). Survival and the four histopathological subtypes (A to D). We usually followed up with thoracoabdominal computed tomography (CT) and whole body bone scintigraphy or positron emission tomography (PET) and tumor marker check at least once a year. The follow-up period for the 96 patients who are alive with or without metastasis ranged from 5 to months (mean, 47.6 months). One patient was lost to follow up. Nineteen patients had metastasis of breast cancer within 39.6 months after initial surgery, and 14 of these patients died of disseminated breast cancer within 72.9 months after surgery. Metastasis of TNIC occurred after a short period and the relapse-free survival time was 4.1 to 39.6 months (mean, 15.9 months); 14 patients had metastasis within 2 years. The site of initial metastasis was the lung in seven patients, lymph nodes in five patients, liver in two patients, bone in one patient, and others in two patients. The relapse rate of type B was the highest (36.4%), followed by type D (20%) (Fig. 4). Disease-free survival (DFS) of type B was shorter than the others, but the difference was not significant (Fig. 6A). Overall survival (OS) of type B was the shortest (P = 0.02) and almost all of the patients with recurrence died within 3 years (Fig. 6B). The reason for the lower OS than DFS rate was that many relapses occurred during the same period and all the disease-relapsed patients died. The type was confirmed by multivariate analysis including histological type (type B versus others, P = 0.01), age (<35 vs 35, P = 0.2), tumor size ( 2.0 cm vs >2.0 cm, P = 0.06) and lymph node metastasis (negative versus positive, P = 0.06). Ishikawa et al. Cancer Sci March 2011 vol. 102 no

5 (A) (A) Fig. 6. (A) Disease-free survival of type B and others. Overall survival of type B and others. Fig. 5. Different immunostaining for estrogen receptor (ER) in ductal components (dcic) and invasive components (iic) of triple-negative invasive carcinoma. (A) High-power view of dcic, immunohistochemistry for ER: tumor cells are positive. High-power view of iic, immunohistochemistry for ER: tumor cells are negative. Table 4. Immunostaining for ER, PgR, HER2 and EGFR in ductal (dcic) and invasive (iic) components of 97 triple-negative invasive carcinomas dcic iic n % ER (+) ()) ()) ()) PR (+) ()) ()) ()) HER2 (+) ()) ()) ()) EGFR (+) (+) ()) (+) ()) ()) Bold value, ER, PR, HER2 and EGFR expressions were not consistent between dcic and iic. ER, estrogen receptor; PgR, progesterone receptor. Discussion The percentage of TNIC with a ductal component was 30% in the present study. It has been reported that the incidence of a ductal component is generally low (18%) among patients with TN breast cancer and that the volume of this component is very small if it is present. (27,28) The incidence of a ductal component in the present study was slightly higher in this series compared with other reports. This might have been because not only specimens of the main section, but also other parts of the tumor were carefully examined. There was a significant difference with respect to the presence of a ductal component, because type A had no ductal component (0 16) and only 18.2% (2 11) of type B had a ductal component, while it was found in 35.9% (23 64) and 60% (3 5) of types C and D. Hormone receptor expression by TNIC was compared between the ductal and invasive components (dcic and iic), and differing patterns of expression were observed in six (6%) out of 29 patients. Estrogen receptor expression was not consistent between dcic and iic in five patients, with dcic being positive and iic being negative in all of them. Four out of the five cases were type C and one case was type D. In addition, we suggested that most of the PR HER2 expression (except for one case, respectively) was similar for both dcic and iic; ER expression sometimes became negative and EGFR sometimes became positive (five and 13 cases, respectively) with the progression from dcic to iic. There are two possible ways for TNIC to develop, that is, cells may be triple negative from the early phase of intraductal proliferation, which leads to invasion, or hormone receptor 660 doi: /j x

6 expression may be lost prior to invasion so that EGFR becomes positive. (18,27,29 31) Based on the concurrent rate of the ductal component, types A and B may be considered to arise due to invasion of cells with TN properties from the initial stage of carcinogenesis, because of the low rate of a ductal component. Because there were only six (6%) cases with non-tn cells in dcic and TN cells in iic and five of the six cases were type C, the former pathway is a minor way of type C. However, only 29 out of 97 patients (30%) had a ductal component in this series, which was not a sufficient sample to assess the mechanism involved. Because EGFR expression was observed in iic more than in dcic, EGFR might have an important role in tumor invasion. P53 positivity was similar with previous reports. (17 19) and it might have some role in triple negative breast cancer (TNBC) carcinogenesis, although there were no differences between histological subtypes and between dcic and iic. It has been suggested that MIB1 initially positively increases in dcic followed by iic, and tended to increase along with tumor invasion. MIB1 positivity in TNDCIS patients has only been assessed in a few studies, (18) and the results of this study were similar. MIB1 positivity of dcic has not been reported before, while MIB1 positivity of iic has varied. Most reports have indicated that the MIB1 index of triple-negative invasive cancer was higher than that of non-triple-negative cancer and ranged around 40 60%. (17,30,32) Comparison of each study shows that positivity increases in the order of DCIS, dcic and iic. This appears to suggest that proliferation increases with the progression of tumor invasion. Our study is unique in analyzing TN breast cancer according to tissue subtypes. As subtypes with a high incidence, atypical medullary carcinoma (AMC, type A) and carcinoma with a central acellular zone (CAZ, type B) were investigated. Atypical medullary carcinoma has been reported to have a 10-year overall survival rate that lies between medullary carcinoma (84 92%) and non-medullary carcinoma (61-63%). (25,33) Atypical medullary carcinoma has sometimes been reported to possess the BRCA1 germline mutation and has often been indicated to have a high incidence among basal-type carcinoma in recent years. (1,12) Review of the prognosis of these tissue subtypes showed that the prognosis is not so bad for type A, although MIB-1 expression is generally high. (12) Most medullary carcinomas and AMC are invasive tumors of grade 3, but the prognosis has been reported to be better than that of conventional grade 3 invasive cancer without inflammation. (34,35) This is considered to be attributable to the host immune response delaying tumor progression and our results did not contradict such a hypothesis. In 1999, Tsuda et al. (15) defined type B as having a CAZ that covered 30% of the entire tumor area. They stated that it has high CK14 positivity and comprises necrotic cells, ischemic tissue, collagen and hyaline material. In the present study, the recurrence rate of type B cancer was 36.4% (4 11), which was higher than the rate of 6.3% (1 16) for type A cancer, 20% (13 64) for type C cancer and 20% (1 5) for type D cancer. The OS was significantly shorter for patients with type B cancer compared with those with types A, C and D. The result was also confirmed by the multivariate analysis. The OS is reported to be worse for patients with grade 3 invasive cancer having a CAZ than for those whose tumors are without a CAZ, and metastasis to the lungs or brain is more likely to occur in the former group. (1,14 16,36) It is classified as having a poor prognosis despite the lack of lymph node metastasis, and fibrosis and necrosis in TN carcinoma is considered to be a risk factor for distant metastasis. A significantly worse prognosis of this group was confirmed by the present study. (4,28) The wide foci of tissue death in CAZ is considered to occur in association with massive infarction involving tumor tissue, both neoplastic and stromal cells, caused by extensive ischemia. Tsuda et al. (15,16) pointed that the reason for the worse prognosis of patients with CAZ was not caused by fibrosis, but by rapid tumor growth of highly anaplastic cancer cells arranged in a solid nest that underwent myoepithelial differentiation, and the large central acellular zone probably developed at an early stage of tumor growth. Hematogeneous metastasis of the CAZ might be related to the process of tumor formation and anaplastic (myoepithelial) differentiation of tumor cells. (15,16) Bone metastasis is reported to be rare compared with involvement of the lungs or brain. (1,2,36) The most frequent site of initial metastasis in the present study was the lungs, followed by the liver, and bone metastasis was only observed in two patients. The lungs were also the site of initial metastasis in the six patients who showed recurrence during this study. Brain metastasis was uncommon, possibly because thoracoabdominal CT and bone scintigraphy were usually used at our hospital to detect recurrence and examination of the brain was not performed until the onset of neurological symptoms, so that it was not classified as the site of initial recurrence. Because TN breast cancer is categorized as ER PR HER2 negative, it includes various tumors. Due to the limited availability of effective therapy, treatment is often difficult and the prognosis can be poor. Although a method that allows screening for tumors with a particularly poor prognosis has not been determined at present, our tissue subtype classification indicated that the prognosis of type B carcinoma was significantly worse compared with that of other types. 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