Int. Med J Vol. 4 No 1 June 2005

DEMOGRAPHIC AND CLINICOPATHOLOGICAL CHARACTERISTICS AND ESTROGEN RECEPTOR STATUS OF BREAST CANCER IN PAHANG DARUL MAKMUR MALAYSIA.

Khurshid Anwar[a] ^b, Falah Salih[b], Tanveer Ahmad Khan[c]^@, M.I.A. Mustafa Mahmud^b, Mohd Maizam^b, Nasser Amjad^b.

ABSTRACT

Breast carcinoma is one of the most common cancers in women. In this retrospective study we evaluated randomly collected 93 cases of breast carcinoma diagnosed in Hospital Tengku Ampuan Afzan (HTAA) Kuantan Malaysia during 1996-2001 for demographic and clinicopathological characteristics, and estrogen receptor (ER) status, using H & E and immunohistochemically stained sections from paraffin blocks. Breast carcinoma was found to be more common in females over age 45 yrs, almost equally distributed between the Malay and Chinese. Most patients were diagnosed at a relatively early clinical stage and were ER positive. ER positivity was more common in the older age, in tumors < 2cm in diameter, in early stages and in tumors with low histological grade. Despite most patients being lost to follow up, ER positivity was found more in patients who were alive for more than 25 months after their initial diagnosis. Moreover, apparently high survival rate of the followed up patients in this series (39 of 46) is probably related to the early diagnosis and relatively high ER positivity rate. Breast carcinoma diagnosis at an early   stage and the frequent positivity for ER opens the door for good prognosis if patients are treated properly.  (Int. Med. J Vol 4 No. 1 June 2005)

Key words:  Breast cancer, Estrogen receptor, Immunohistochemistry.

INTRODUCTION

Breast cancer is the most common cancer among women, excluding non-melanoma skin cancers, and the second leading cause of cancer deaths in women today lagging only behind lung cancer (1, 2). There is a strong inherited familial risk of breast cancer in some families and recent data reveals up to 27% of breast cancers may be attributed to inherited factors and mutated BRCA1 and BRCA2 genes are responsible for approximately 30-40% of inherited breast cancers (3). Some racial groups have a higher risk of developing breast cancer, notably; women of North American and European descent have been noted to have a higher rate of breast cancer than women of African and Asian origin (4). According to the second report  of the Malaysian National Cancer Registry in 2003, breasts cancer was the most common cancer in all ethnic groups and comprised 30.4% of all newly diagnosed cancer cases among Malaysian women in 2002 (5).

Risk factors for breast cancer include, increasing age, genetics (BRCA1 & BRCA2), personal and family history of breast cancer, previous history of benign breast conditions, prolonged reproductive period, delayed childbirth and nulliparity, obesity, high fat diet, previous radiation therapy and hormonal replacement therapy beside smoking and daily consumption of alcohol (6).

Histological type and grade of cancer, tumor size, lymph node metastasis, stage and certain immunohistochemical makers like estrogen receptor (ER)/progesterone receptor (PR) status and CerbB2 have consistently been shown to be important prognostic factors for breast cancer survival. Poorer prognosis is associated with larger, higher grade tumors, with axillary lymph node involvement, distant metastasis, negative hormone status and positive C-erbB2 staining (7).

Recently published scientific data indicates that ovarian hormones, principally estrogens, play a major role in the etiology of breast cancer by affecting the rate of breast epithelial cell proliferation, perhaps via stimulation of the expression of genes encoding for growth factors (8, 9, 10).  Intracellular ERs bind and transfer estrogen to the nucleus, where it interacts with estrogen response elements on DNA, thereby activating nearby target genes and resulting in the synthesis of proteins involved in cell division (11, 12). Although ERs exist in normal breast epithelial cells to regulate breast development during puberty and pregnancy, they are usually present in extremely low quantities (13, 14). In contrast, 30% of premenopausal and 60% of postmenopausal breast cancers have measurable ERs (15, 16). Generally, tumors expressing these receptors tend to respond more favorably to hormonal therapies and have a better overall outcome than tumors not expressing ERs or PRs (17).  

The purpose of our project of which this is the first publication was to collect and correlate demographic and clinicopathological data of patients with breast cancer diagnosed in Pahang Darul-Makmur Malaysia from 1996 to 2001 with a series of immunohistochemical markers to evaluate their potential  as prognostic markers.

In this paper we are going to present the salient demographic and clinicopathological characteristics of breast cancer patients and correlate them with ER status of their tumors.

MATERIALS AND METHODS

Patients:

The study included 100 randomly collected histologically confirmed breast carcinoma cases from the files of the Pathology Department of Hospital Tengku Ampuan Afzan (HTAA), Kuantan, Pahang, D.M. Malaysia, diagnosed during 1996-2001. The criterion for inclusion was determined by the availability of sufficient tissue for H & E (Hematoxylin and eosin) staining and further immunohistochemical studies. Based on theses criteria  93 cases were included in the present study. The available demographic and clinicopathological data were collected from the biopsy request forms or patient’s files wherever possible. However, for some patients we couldn’t obtain sufficient relevant information due to patient default, incomplete entry of data in patient’s files or their referral to another center for treatment. At least two blocks of the tumor tissue from each patient were analyzed to reconfirm the original histological diagnosis and to determine the histological grade. Tumors were classified in accordance with NHSBSP guidelines (18) and histological grading was established using modified Bloom's grading system described by Elston and Ellis (19). The tumors were staged according to the American Joint Committee on Cancer staging system, grouping patients based on the tumor size (T), lymph node status (N), and distant metastases (M) into 4 stages, thus allowing clinicians to derive prognostic information necessary for therapeutic decisions (20).

Immunohistochemistry:

Formalin-fixed paraffin sections of breast cancer tissue were mounted on glass slides coated with 3-aminopropyl-triethoxysilane (APES; Sigma, Poole, Dorset, UK) and were baked for 30 min at 56–60°C, before being dewaxed in xylene. The tissue sections were rehydrated by sequential immersion in 100% and 50% ethanol to distilled water and then subjected to heat antigen retrieval for 40 min in citrate buffer (pH 6) in a jar containing preheated (95-99 ⁰C) target retrieval solution. After cooling, tissue sections were incubated for 5 min in 0.3% (v/v) hydrogen peroxide. Subsequently, the sections were washed in tap water and Tris-Buffer (pH 7.45) and were exposed to normal rabbit serum (diluted with Tris) for 30 min at room temperature (20–24°C). Diluted primary antibody (anti-ER) was applied and incubated overnight at 4°C (18 hours). After washing with Tris, biotinylated rabbit anti-goat secondary antibody, together with the Strept-AB Complex/HRP (0377, DAKO, Glostrup, Denmark) was applied for 30 min at room temperature. Staining was revealed by development in the chromogen 3, 3-diaminobenzidine tetrahydrochloride (DAB) for 5–30 min. Sections were rinsed with DW and mounted using cover slip with aqueous bases mounting medium. The intensity of immunostaining was labeled as +, less than 25% of cells stained intensely, ++, 25-50% of cells stained intensely, +++, 51-75% of cells stained intensely and ++++, more than 75% of cells stained intensely. The pattern of staining varied (nuclear, cytoplasmic, membranous) depending on the type of antibody used.

RESULTS:

Demographic data:

The mean age of the patients included in the study was 51.3 ranging between 32 and 89 year old. The majority  of the patients (60%) were  41-60 years of age (Table 1).

Table 2 summarizes the demographic and some of the clinicopathological  characteristics of the 93 breast carcinoma cases included in this study. The ethnic distribution was comparable in Malays and Chinese constituting 50% and 44% of the total cases respectively. The Indian constituted only 6.5%. The most frequent histological type of breast cancer (93.6%:87/93 cases) in this study was infiltrating ductal carcinoma. The others were two non-invasive ductal carcinoma, two papillary carcinoma and one each medullary and mucinous carcinoma. Most of the patients (57%) had tumor size ranging between 2-5 cm in diameter (T2) and lymph node metastasis was observed in 50.5% of cases while distant metastases was observed in only 2 cases. At the time of presentation 71% of the patients were in stage 2 and 60% had histological grade 2  tumors. Most of the patients (64.5%) presented with  right sided tumors.

Regarding treatment all patients had undergone surgical treatment alone or in combination with adjuvant modalities like radiotherapy, chemotherapy (Table 3). Only 37.6% (35/93) of the patients received tomoxifen treatment, while records for the other 58 cases did not reveal any hormonal treatment.  

Nearly half of the patients (47) were lost to follow up, 27 were alive with no recurrence, 12 alive with recurrence and 7 were dead by the end of follow up period in July 2004 (Table 4).

21 patients were alive after 48 months of the diagnosis, 25 patients were alive for 25-48 months since their first diagnosis, 15 had only 12-24 months follow up period and 11 patients had less than  one year follow up period (Table 5).

Information about parity of the patents were available for only 44 cases, 19 of these were nulliparous, 14 had 1- 3 children and rest of 11 had more than 3 children. Family history of cancer was available for 45 cases, 4 cases had family history of breast cancer, 3 cancers other than breasts and rest of 38 had no family history of any cancerous lesion. Out of 43 cases with information about prior hormonal treatment only seven had history of hormonal treatment in past few years. Eight cases were unmarried out of 44 for which marital status was known.

ER status of tumors

Most of the breasts cancer tissues (54.8%) were found positive for ER. The intensity and extent of  staining in tumor tissue is graded as mentioned in material and methods (Table 6). Nuclear staining was positive in all cases, however 38 cases revealed variable degree of cytoplasmic staining too (Figures 1-3).  

Table 7 shows status of ER in relation to some of the demographic and clinicopathological characteristics of the tumors. ER positivity was more common in patient over 45 year of age rather than in patients below 45(37/63 vs. 14/30). Both non-infiltrating ductal carcinoma and 3 of the 4 other types were positive for ER. Most of the tumors of less than 2 cm size (T1), (12/14) were positive for ER. Tumors with or without axillary lymph node metastasis had similar rate for ER positivity. 43 of 77 low stages (1 & 2) were positive for ER compared to 8 of 16 high stage tumors. More of low histological grade (G1) tumors (13/19: 68.4%) stained with ER antibody than the high histological grade (G2 & G3) ( 38/74: 51.4%).

25 of 39 cases with localized disease and 3 of 7 dead cases were ER positive (Table 8). Most of the cases with  25-48 months survival period (19/25: 76%) and those with more than 48 months survival  (12/21:57.1%) were positive for ER (Table 9).

Figure 1. Immunohistochemical staining of estrogen receptor in an

infiltrating ductal carcinoma (20 HPF)

Figure 2. Immunohistochemical staining of estrogen receptor in an

infiltrating ductal carcinoma (40 HPF)

Figure 3. Immunohistochemical staining of estrogen receptor in an

infiltrating ductal carcinoma (100 HPF)

DISCUSSIONS

Breast cancer risk increases markedly after 40 year of age. A women’s chance of breast cancer increases from one out of 235 at age 40 to one in 54 at age 50 and then it continues to increase further with increasing with age (21). In the present study 78 of 93 cases were in age group above 40. The Second report of Malaysian National Cancer Registry shows that most of  breast cancers occurred in age group 40-60(5), which is comparable to our results  as 57/93 cases ( 61.3%) were in the same age group in our study.

Ethnic distribution of breast cancer was almost same as that reported in Malaysian National Cancer Registry report (5) and both Malay and Chinese patients had comparable distribution with slight predominance of the former, however, Indians were underrepresented in our study and constituted only 6.5% of total cases.

The predominant histological type of breast carcinoma in this study was infiltrating ductal carcinoma as reported in most of other studies (20).

Most of the patients ( 72%) presented with tumor size <5 cm in diameter (T1-T2), while 46 of 93 cases (49.5%) had no metastasis to lymph nodes and only two cases were confirmed to have distant metastatic spread. These findings suggest that most of the patients with breast cancer in our series presented at an early stage of the disease where 77 of 93 cases (82.8%) were either in Stage 1 or Stage 2.  This indicates the efficacy of the breast cancer awareness programs carried out by the NGOs and Malaysian government agencies (22, 23).

Histologically most of the breast cancers were either in Grade 1 (20.4%) or Grade 2 (60.2%), once again indicating relatively good prognosis. Previous studies have confirmed the histological grade association with prognosis and chemotherapeutic response (19, 24)

Interestingly most of cases (64.5%) had right sided breast cancer in contrary to various other reports where left sided cancer was the commonest (25, 26). It has been suggested that in about 55% of women, the left breast is slightly larger than the right and therefore contains a larger amount of tissue at risk for becoming cancerous (26). It will be interesting to evaluate this hypothesis regarding the increased incidence of right sided breast cancer in our patients. However, this discrepancy may be due to the limited number of cases in the present study.

Most of the patients were treated with only surgery (49 of 93 cases), most probably due to their presentation in early stages. The rests of the cases were additionally treated with other adjuvant modalities such as chemotherapy and radiation.

Survival data and follow up of patients was not maintained according to the standard practice, as 50% of cases were lost to follow up after a few visits to hospital. However, 46 patients had follow up data available for more than 25 months and 21 of them for more than 48 months after their first diagnosis. Only 7 cases were confirmed to be dead in this series by July 2004 (the last date of follow up). Although longer follow up periods are required (at least 5 years), our data collectively suggest a better prognosis in this series of patients which we believe is due to the earlier stage at the time of presentation and also probably due to the appropriate treatment these patients received.

Thirty three of 44 cases (75%) with breast cancer were either nulliparous or had 1-3 children which indicates that reduced fertility is one of the common associations with breast cancer as shown in previous studies(6, 27). Only 4 out of 45 had family history of breast cancer which is in confirmation with the previous data of familial breast cancer representing 5-10% of total breast cancers (6, 28), however, only 7 of 43 cases had prior history of any hormonal treatment in our study.

There is substantial scientific data indicating that breast carcinomas expressing estrogen receptors have better prognosis because they are more differentiated and favorably respond to hormonal treatment compared with ER negative tumors (17, 29-30). Moreover, patients with ER positive tumors have prolonged disease free survival after primary treatment, superior overall survival and longer survival after recurrence compared with patients having ER-negative tumors, which is independent of axillary node status (29). 54.8% (51/93) of the tumors in this study were positive for ER especially those with tumor size less than 2 cm in diameter (T1). Moreover, ER positivity was significantly more in stage 1 and 2 compared to stage 3 and 4 (p<0.05). Most of the cases having follow up for 25-48 months or more than 48 months were also ER positive and only 3 of 7 patients who died during our study period had  ER positive tumor. This clearly indicates the good prognosis of tumors with positive ER status.

CONCLUSION

In conclusion our study shows that most of the breast cancer patients attending the breast clinic in HTAA Kuantan, present at an early stage and are ER-positive, thus having good prospect for favorable  prognosis if  appropriately treated.

ACKNOWLEDGEMENT

We thank Dr. Sunita Ahlawati, Dr. R. Kalavathy and Mr. Bakhta of Pathology department of HTAA for providing us pathological material, Ms Juriah for her expert technical help in immunohistochemical staining and Dr. Azmi, Dr. Jefri Bin Din, Dr. Joe and Dr. Herbajan Singh of surgical unit of HTAA Kuantan for permitting us to include their cases for the present study.This study was supported by the Research Centre and the Faculty of Medicine, International Islamic University of Malaysia (IIUM), Kuantan, Pahang, Malaysia. 

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Tables

Table 1. Age Distribution of 93 Breast Carcinoma Cases from HTAA Kuantan During 1996-2001.

Table 2. Demographic and Clinicopathological Characteristics of 93 Breast Carcinoma Cases

from HTAA Kuantan During 1996-2001.

 Table  3. Type of Treatment Received by 93 Breast Carcinoma Cases

 from HTAA Kuantan During 1996-2001.

Table  4.  Clinical outcome of 93 Breast Carcinoma Cases from HTAA Kuantan During 1996-2001.

Table  5. Follow up Data of 93 Breast Carcinoma Cases from HTAA Kuantan During 1996-2001.

Table  6. Intensity and Extent of ER staining in 93 Breast Carcinoma Cases from

HTAA Kuantan during 1996-2001.

Table 7. Association of Estrogen Receptor (ER) Status with Demographic and Clinicopathological Characteristics

of 93 Breast Carcinoma Cases from HTAA Kuantan During 1996-2001.

Table  8. Association of Estrogen Receptor (ER) Status with Clinical outcome of 93 Breas

Carcinoma Cases from HTAA Kuantan During 1996-2001.

Table  9. Association of estrogen receptor (ER) status with follow up data of 93 breast carcinoma

cases from HTAA Kuantan during 1996-2001.

[c] Faculty of Pharmacy, International Islamic University Malaysia (IIUM), 25200 Kuantan, Pahang, D.M. Malaysia.