During the five years under study, all 458 samples that fulfilled the inclusion criteria were analysed. The age range of patients with breast carcinomas was 23 to 85 years with a mean age of 48 ± 12.03 years and a median age of 46. All the breast tumours were female and the disease was most common among 41-50 age group (34.4%) followed by 31-40 (26%) while the least values of 3.6% and 1.1% were recorded for 21-30 and 81-90 respectively (Table 1).
Mastectomy specimens were 137 while biopsy specimens were 321, and majority of the tumours have sizes ranging from 2-5cm in 39.9% of the cases. Invasive ductal carcinoma, NOS (not otherwise specified) dominate as the commonest histological subtype in 91.5% of cases.
The tumours were of grade II and grade III in 48.5% and 41.0% of cases respectively at the time of diagnosis. Fifty-seven (57) mastectomy specimens have accompanying lymph nodes; they display metastases in 84.2% of cases. The tumours were predominantly triple negative; ER, PR, and Her-2/neu were negative in 93.2%, 97.8%, and 95.6% of cases respectively (Table 2).
Correlation of age with ER/PR status shows that there were 16 cases in age bracket 21-30, out of which 15 (93.7%) were seen to exhibit ER-PR-. One hundred and six (92.2%) of cases in the age group 31-40 had ER-PR-, followed by 5(4.3%) for ER+ PR- while only 1 (0.9%) was recorded for ER + PR +. It was discovered that ER- PR- has the highest percentage of all the age groups followed by ER+PR- while ER-PR+ was uncommon for all ages. P value of 0.955 indicates that there is no relationship between age group and hormone receptor status (as ER-PR- inconsistently dominates all age groups) (Table 3).
24
The correlation of hormone receptor status and size of tumour shows that 124 cases were less than 2cm in size. Thirteen (10.5%) of the cases were ER +PR-, 1(0.8%) was ER- PR+ while the largest value of 109 (87.9%) was recorded for ER- PR-. Of the 169 cases that were between 2-5cm, 153 (90.5%) were observed to be ER-PR-, 4 (2.4%) were recorded for ER-PR+, 10 (5.9%) were ER+PR- while 2 (1.2%) were ER+PR+. P= 0.03 rejects the null hypothesis of no relationship and therefore shows that hormone receptor status depends on size of the tumour. This association is due to ER- PR- which is dominant and increases with increase in size ,that is 87.9%
, 90.5% and 97.7% for <2cm, 2-5cm and >5cm respectively (Table 4).
Table 5 shows the number and percentage of hormone receptor status seen in each histological type. Invasive ductal carcinoma, not otherwise specified (IDC,NOS) was the most common, out of which ER-PR- had the highest value of 91.6%, followed by 6.2% for ER+PR- while 1.4 and 0.7% represented ER-PR+ and ER+PR+ respectively. Eighty percent of mucinous, 100% papillary and 100% medullary were also ER-PR-. Of the Invasive Lobular Carcinoma (ILC) 95.5% was ER-PR- while just 1 (4.5%) was ER+PR-. All the cases of comedo-carcinoma seen were ER-PR-. P = 0.798 shows that there is no relationship between histological type and hormone status.
Table 6 reveal the correlation between histological grade and hormone receptor status. It was discovered that ER-PR- dominates all the age groups, representing 89.6% for grade 1, 91.9% for grade II and 92% for grade III. P value of 0.700 indicates that there is no relationship between these two parameters, that is, tumour grade does not determine the hormone receptor status of the tumour.
25
Her-2/neu- was 97.9% for grade I, 94.1% for grade II and 96.8% for grade III while Her-2/neu+
was 2.1% for grade I, 5.9% for grade II and 3.2% for grade III. P value of 0.505 shows no relationship, that is, grade does not determine the Her-2/neu status (Table 7).
Table 8 shows correlation between lymph node metastases and hormone receptor status. A P value of 0.174 indicate that hormone receptor status is independent of lymph node metastases.
Table 9 shows IHC positivity according to histological grade. Oestrogen receptors positivity decreases with increase in the grade of the tumour. Histological grade has variable relationship with progesterone and Her-2/neu receptors positivity.
Table 10 shows association of Her-2/neu status and ER, PR expression. Oestrogen and progesterone receptors negativities are closely related to Her-2/neu reactivity.
Co-expression of ER, PR reveal positivity in 3 cases (0.7%) while ER/PR was negative in 420 cases (91.7%), (Figure 1).
Figures 2-4 show the sections of ER, PR, and Her-2/neu positive stained cells.
26
TABLE 1-THE DISTRIBUTION OF PATIENTS ACCORDING TO AGE GROUPS.
Age group Number of Cases (%)
21-30 16 (3.6)
31-40 115 (26.0)
41-50 152 (34.4)
51-60 84 (19.0)
61-70 54 (12.2)
71-80 16 (3.6)
81-90 5(1.1)
27
TABLE 2- THE CLINICO-PATHOLOGICAL FEATURES AND RECEPTOR STATUS Histopathological features Number of cases (%)
TYPE OF BIOPSY Incisional Biopsy Excisional Biopsy Mastectomy Trucut
114 (24.9) 39 (8.5) 137 (29.9) 168 (36.7) TUMOUR SIZE
<2 cm 2 – 5 cm
>5 cm Total
124 (29.2) 169 (39.9) 131 (30.9)
424 HISTOLOGICAL TYPE
IDC, NOS ILC Mucinous Papillary Medullary
Comedo Carcinoma
419 (91.5) 22 (4.8) 10 (2.2) 2 (0.4) 1 (0.2) 4 (0.9) HISTOLOGICAL GRADING
I II III
48 (10.5) 222 (48.5) 188 (41.0) LYMPH NODE METASTASIS
Present Absent Total
48 (84.2) 9 (15.8)
57 ER
Positive Negative
31 (6.8) 427 (93.2) PR
Positive Negative
10 (2.2) 448 (97.8) HER 2/NEU STATUS
Positive Negative
20 (4.4) 438 (95.6)
28
TABLE 3- CORRELATION OF AGE WITH ER/PR STATUS
Age group
Number of Cases and Percentage
ER+ PR+ (%) ER+ PR-(%) ER- PR+ (%) ER- PR-(%)
21-30 16 (3.6%) - 1 (6.3) - 15 (93.7)
31-40 115 (26.0%) 1(0.9) 5 (4.3) 3 (2.6) 106 (92.2) 41-50 152 (34.4%) 1(0.7) 7 (4.6) 4 (2.6) 140 (92.1)
51-60 84 (19.0%) - 8 (9.5) - 76 (90.5)
61-70 54 (12.2%) 1 (1.9) 4 (7.4) - 49 (90.7)
71-80 16 (3.6%) - - - 16 (100)
81-90 5 (1.1%) - - - 5 (100)
29
TABLE 4 - SIZE VS HORMONE RECEPTOR STATUS
Size Number of cases and percentage
ER+ PR+ (%) ER+ PR-(%) ER- PR+ (%) ER- PR-(%)
<2 cm 124 (29.2%) 1 (0.8) 13 (10.5) 1 (0.8) 109 (87.9) 2 – 5 cm 169 (39.9%) 2 (1.2) 10 (5.9) 4 (2.4) 153 (90.5)
>5 cm 131 (30.9%) - 2 (1.5) 1 (0.8) 128 (97.7)
30
TABLE 5 - HISTOLOGICAL TYPE VS HORMONE STATUS Type of
tumour
Number of cases and percentage
ER+ PR+ (%)
ER+ PR -(%)
ER- PR+ (%)
ER- PR-(%)
IDC, NOS 419 (91.5%) 3 (0.7) 26 (6.2) 6 (1.4) 384 (91.6) Mucinous 10 (2.2%) - 1 (10.0) 1 (10.0) 8 (80.0)
Papillary 2 (0.4%) - - - 2 (100.0)
Medullary 1 (0.2%) - - - 1 (100.0)
ILC 22 (4.8%) - 1 (4.5) - 21 (95.5)
Comedo Carcinoma
4 (0.9%) - - - 4 (100.0)
31
TABLE 6- GRADE VS HORMONE RECEPTORS Grade Number of cases and
percentage
ER+ PR+ (%) ER+ PR-(%) ER- PR+ (%) ER- PR-(%)
I 48 (10.5%) 2 (4.2) 2 (4.2) 1 (2.1) 43 (89.6)
II 222 (48.5%) - 15 (6.8) 3 (1.4) 204 (91.9)
III 188 (41.0%) 1 (0.5) 11 (5.9) 3 (1.6) 173 (92.0)
32 TABLE 7- GRADE VS HER-2/NEU
Grade Number of cases and percentage HER-2/NEU- HER-2/NEU+
I 48 (10.5%) 47 (97.9) 1(2.1)
II 222 (48.5%) 209 (94.1) 13 (5.9)
III 188 (41.0%) 182 (96.8) 6 (3.2)
33
TABLE 8- LYMPH NODE METASTASES VS HORMONE RECEPTORS Lymph
nodes metastases
Number of cases and percentage
ER+ PR+ (%) ER+ PR-(%) ER- PR+ (%) ER- PR-(%)
Present 46 (84.2%) - 1 (2.1) 1 (2.1) 46 (95.8)
Absent 9 (15.8%) - - - 9 (100.0)
34
TABLE 9- IHC POSITIVITY ACCORDING TO HISTOLOGICAL GRADE.
Grade Cases ER+ PR+ HER-2/NEU+
I 48 4 (8.33) 3 (6.25) 1 (2.08)
II 222 15 (6.76) 3 (1.35) 13 (5.86)
III 188 12 (6.38) 4 (2.13) 6 (3.19)
35
TABLE 10- ASSOCIATION OF HER-2/NEU STATUS AND ER AND PR EXPRESSION
HER-2 TOTAL ER- ER+ PR- PR+
Negative 438 (95.6) 409 (93.68) 29 (6.62) 428 (97.72) 10 (2.28) Positive 20 (4.4) 18 (90.0) 2 (10.0) 20( 100.0) - Total 458 (100.0) 427 (93.23) 31 (6.77) 448 (97.82) 10 (2.18)
36
FIGURE 1-THE HORMONE RECEPTOR STATUS OF THE TUMOURS
37
FIGURE 2- SECTION SHOWING IHC-OESTROGEN RECEPTOR ANTIGEN STAINED CELLS REPRESENTED IN BROWN AREAS(X40).
38
FIGURE 3-SECTION SHOWING IHC-PROGESTERONE RECEPTOR STAINED CELLS REPRESENTED BY BROWN AREAS(X40).
39
FIGURE 4-SECTION SHOWING IHC- HER-2/NEU RECEPTOR STAINED CELLS REPRESENTED BY BROWN AREAS(X40).
40 CHAPTER FIVE
DISCUSSION
The study shows that the mean age at diagnosis is 48 + 12.03 years. This is in agreement with studies done in black Africans2. This contrasts with the age at diagnosis in Caucasians of 60 years2. Population demographics and genetic variation may account for the differences62. Furthermore, breast carcinomas are common in premenopausal Africans compared to Caucasians with a majority of breast carcinomas occurring in postmenopausal women. The average age at menopause in Africans varies between 46 and 49 years as against an average of 50 years Worldwide73-75. Higher incidence of premenopausal breast cancer in Africans is related to population demographics. The life expectancy in Africans is very much lower than Caucasians62. Environmental factors may be related62.
Invasive ductal carcinoma (IDC), NOS is commoner than invasive lobular carcinoma (ILC). In this study, IDC accounts for 91.5% as against ILC of 4.8%. Worldwide IDC predominates and Li, Uribe and Daling reported a value of 75% for IDC and 15% for lobular carcinomas for all cases in the United States of America 3.
More than 30% of our patients presented with large tumour size greater than 5.0 cm and in grade II (48.5%) and grade III (41.0%). This agrees with results found in other black African countries 6. Late presentations and aggressive tumour biology are reasons for large breast tumours of higher grade in African women6,7,8.
The tumours in this study are predominantly triple hormone receptor negative. Similar observations were made by several studies done amongst Africans2, 60. Oestrogen receptor
41
reactivity was 6.8% which was comparatively lower than two studies done in Nigeria, which both reported 25%22 , 59.
The relevance of this, is that some anti-oestrogens and anti-hormonal drugs targeting breast carcinomas may be less effective in many Africans.
Large-sized tumours display negative hormonal receptor status in this study. This is consistent with the study done by Fatima et al in Karachi, Pakistan 76. Non-reactivity was found to have increased with high grade tumours on correlation of ER/PR hormonal status with grading. This observation is similar to that of Suvarchala et al54.
Co-expression of hormones is said to be important in the pattern of metastases of breast tumours and responsiveness to treatment. Negativity for ER and PR (ER-PR-) was found to dominate all age groups and increases with large tumour sizes as well as the grade of the tumours. Furthermore ER +PR+, ER +PR- and ER-PR+ have inverse relationship with grade of tumour. This is in keeping with observations made by Suvarchala et al and Fatima et al in India and Pakistan respectively 54 , 76.
Her- 2/neu positivity also correlates with higher grade tumours in this study. This observation is similar to that of Azizun-Nisa et al.
Finally, it is important to note that no correlation between lymph node metastases and hormone status positivity is found.
42 CHAPTER SIX CONCLUSION
This study highlights the fact that breast carcinomas in Africans seem to occur in younger age and they present with high histological grade, large tumour size, and more often triple negative hormonal status. Therefore, there is tendency towards poor response to hormonal therapy and poor prognosis. This finding agrees with most results in some parts of the country2,58,59. Immunohistochemical evaluation of ER, PR and HER-2/NEU in all breast carcinomas is recommended so that patient’s treatment can be individualised. Centres without facilities for immunohistochemistry should refer patients to Institutions where such tests can be done.
Furthermore, breast cancer screening programme should be strengthened such that non-palpable breast cancers can be detected very early.
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52 APPENDIX i
Protocol for Staining Formalin- Fixed, Paraffin Embedded Tissue Sections Buffer and Reagent Preparation
Peroxidase Quenching Solution
To prepare 200mls of Quenching Solution:
30% hydrogen peroxidase 3mls
Methanol 200mls
Total volume 203mls
*use within 4hours of preparation.
Epitope Retrieval Buffer, Reduced pH
To prepare 200mls of Epitope Retrieval Buffer Working solution:
Concentrated Epitope Retrieval Buffer, 4mls
Reduced pH
Distilled Water 196mls
Total volume 200mls
pH should be 5.8-6.2. Adjust pH, if necessary, with IN HCL or IN NaOH OR
To prepare 1 litre of Epitope Retrieval Buffer Working Solution:
Concentrated Epitope Retrieval Buffer, 20mls
Reduced pH
53
Distilled Water 980mls
Total volume 1000mls
pH should be 5.8-6.2. Adjust pH if necessary with In HCL or IN NaOH.
*Store at to 2-80C. Discard after 3 months.
IHC Wash Solution
To prepare 1 litre of working IHC Wash Solution:
Concentrated IHC Wash solution 5mls
Distilled Water 995mls
Total volume 1000mls
Anti- Rabbit IHC Antibody
Using the dropper bottle provided, prepare Working anti- Rabbit IHC Antibody for 10 slides:
Concentrated anti-Rabbit IHC Antibody 1 drop
Ready-to-use Antibody Diluent 2mls
Total volume 2mls
*Rinse dropper bottle with di-H20 after each use.
DAB Substrate
Using the dropper bottle provided, prepare Working DAB Substrate:
DAB Solution A 1 drop
DAB Solution B 1 drop
DAB Solution C 1 drop
Distilled Water 2.5mls