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ORIGINAL ARTICLE
Year : 2016  |  Volume : 53  |  Issue : 1  |  Page : 34-38
 

Significance of prognostic indicators in infiltrating duct carcinoma breast: Scenario in developing country


Department of Pathology, JSS Medical College, JSS University, Mysore, Karnataka, India

Date of Web Publication28-Apr-2016

Correspondence Address:
J Krishnamurthy
Department of Pathology, JSS Medical College, JSS University, Mysore, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0019-509X.180834

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 » Abstract 

Context: Carcinoma of the breast is one of the most common malignant tumors and is the most common cause of death from cancers in females. Early diagnosis and assessing the prognosis for each patient is essential for a better therapeutic plan and management. Aims: To evaluate the significance of various prognostic indicators of breast carcinoma by correlating with Nottingham modification of Scarff Bloom-Richardson's grading system (NMBGS). Materials and Methods: Eighty four patients who underwent mastectomy for breast carcinoma at a tertiary care centre in South India over a period of 2 years have been evaluated to note the importance of the various prognostic factors correlating them with NMBGS. Statistical Analysis: A Chi-square test was used to determine possible association between the various prognostic factors. Results: Eighty percent of the tumors were infiltrating ductal carcinoma (IDC), and it is seen that the larger tumor size, higher histopathological grade, increased lymphovascular invasion, lymphnode metastasis, tumor necrosis, microvessel density, estrogen and progesterone receptor negativity, and HER-2/neu positivity were associated with higher grade of tumor. Conclusions: The traditional morphological factors including the histological type, grade, tumor size, lymphovascular invasion, lymph node status, presence of necrosis, stromal reaction, and microvascular density (MVD) count are relatively simple but robust prognostic factors to assess, while the hormonal and genetic status not only have prognostic value but are useful predictive marker for adjuvant chemotherapy. Hence, the status of these various prognostic factors should form the basis of all routine histopathological reports in cases of breast cancer for better management.


Keywords: Breast carcinoma, prognostic indicators, Scarff Bloom-Richardson grading system


How to cite this article:
Krishnamurthy J, Kumar P S. Significance of prognostic indicators in infiltrating duct carcinoma breast: Scenario in developing country. Indian J Cancer 2016;53:34-8

How to cite this URL:
Krishnamurthy J, Kumar P S. Significance of prognostic indicators in infiltrating duct carcinoma breast: Scenario in developing country. Indian J Cancer [serial online] 2016 [cited 2020 Jun 6];53:34-8. Available from: http://www.indianjcancer.com/text.asp?2016/53/1/34/180834



 » Introduction Top


Carcinoma of the breast is one of the most common malignant tumors and is the most common cause of death from cancers in females.[1] A woman who lives to the age of 90 has a one in eight chance of developing breast cancer.[2] In the West, widespread use of mammography has resulted in a marked increase in early detection of breast carcinoma and a fall in mortality with appropriate therapy. However, in developing countries, breast cancer mortality is still rising.[1]

The role of histopathology in the provision of prognostic information for a number of tumors has been well-established. Until recently, the main role of the diagnostic histopathologist lay only in the establishment of a basic diagnosis of breast cancer with a note on locoregional lymph node involvement. In recent years with the advent of breast screening for an early detection, it is imperative to assess various prognostic factors for each patient for a better therapeutic plan.[3] Earlier the treatment of breast cancer was predominantly surgical, however, in the last two decades the treatment of breast cancer has undergone dramatic changes and a much wider range of therapeutic options are now available. Thus, it has become increasingly important to assess prognosis for each of these patient before a therapeutic plan is agreed upon.[3]

The number of tumor-related features available to predict the prognosis of patients with breast cancer has grown impressively in recent years. Histology, tumor stage, and lymph node status are now supplemented with measurements of steroid hormone receptors, ploidy, S-phase fractions, growth factors, oncogenes, and oncogenic products. These cellular and molecular biological features have not only advanced the understanding of carcinogenesis, but have provided a host of new biologic measures potentially related to the clinical outcome. The various prognostic indicators not only predicts the disease outcome but, also contributes to the therapeutic planning of patients with breast cancer.[4] Prompted by these, the authors have carried out this study to evaluate the significance of the various prognostic indicators in patients with breast carcinoma.


 » Materials and Methods Top


Eighty four patients who underwent mastectomy for carcinoma breast at a tertiary care center in South India over a period of 2 years from June 2009 to May 2011 have been included in the study.

Following gross examination of the mastectomy specimens in which the tumor sizes were noted the specimens were fixed in 10% formalin, routinely processed, embedded in paraffin and the hematoxylin and eosin-stained sections were studied. The histological types were noted and the invasive ductal carcinomas (IDC) were graded based on Nottingham modification of Scarff Bloom-Richardson grading system (NMBGS).

Various prognostic factors including the tumor size, histological grade, lymphovascular involvement, lymphnode status, tumor necrosis, stromal reaction, microvessel density count, deep resected margin status and nipple and areola involvement were noted.

Immunohistochemical staining was done in 50 cases to evaluate the estrogen receptors (ER), progesterone receptor (PR) and epidermal growth factor (HER-2/neu) status.

Microvessel density (MVD) count was determined by two independent observers. Each evaluated the slides without the knowledge of the counts performed by the other.A single microvessel was defined as any vessel with a clear lumen. The screening of the slides was done by hotspot method in which the slides were initially screened in low power (×10 objective lens, ×10 ocular lens) to identify the areas with highest number of micro vessels or vascularity. The micro vessel count was then performed in these sufficiently extended fields under high power (×40 objective lens, ×10 ocular lens). The mean value of ten most vascularized areas at ×400 field was considered as MVD for the sample.

In cases of ductal carcinomas and mixed carcinomas, which had ductal component the prognostic factors, were correlated with grades to note their significance.

Statistical analysis

A descriptive analysis was made of the variables included in the study. Percentages were calculated for qualitative variables and the mean and standard deviation for quantitative variables.A Chi-square test was used to determine possible association between the various prognostic factors.A significance level of P < 0.05 was used in the statistical analyses. Data was analyzed using the EpiInfo software, version 3.5.3 for Windows.


 » Results Top


In the present study of 84 patients who were diagnosed with breast carcinoma only one was a male and only one patient presented with bilateral involvement. The age group of patients ranged from 28 years to 82 years, with a mean age of 54.89 years and a median age of 55 years. Maximum numbers of patients were in the age group of 50-59 years and accounted for 22.6% of cases. Based on the specification of the TNM staging of tumor size, it was noted that the maximum number of cases (72.9%) belonged to T2 category.

Histologically, the carcinomas were of varying types. The most predominant type was that of invasive ductal carcinoma (80%), while the rare types included metaplastic carcinoma (1.2%), inflammatory carcinoma (1.2%), and angiosarcoma (1.2%). The study included three cases each of mucinous carcinoma (3.5%) and mixed type (3.5%), while there were two cases of medullary carcinoma (2.4%) and five cases of invasive lobular carcinomas.

The ductal carcinoma (68 cases) and the mixed carcinomas (three cases) that had ductal componentwere graded based on NMBGS, which depends on the percentage of tubule formation, nuclear pleomorphism, and the number of mitoses. There were 50.7% cases of grade III, while grade I and II tumors accounted for 2.8% and 46.5%, respectively [Table 1] and [Figure 1]a-[Figure 1]c.
Table 1: Distribution of tumors according to histological grade

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Figure 1: (a) Grade I IDC – Greater than 75% of tumor cells show tubule formation with minimal nuclear atypia and occasional mitoses (H and E, ×100); (b) Grade II IDC – Less than 75% of tumor cells are showing tubule formation with moderate nuclear atypia and 10-12 mitoses/10 HPF (H and E, ×100); (c) Grade III IDC – Tumor cells are arranged in sheets with less than 10% cells showing tubule formation along with marked nuclear atypia and frequent mitoses (>/10HPF) (H and E, ×100)

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In addition, 34.1% of cases showed lymphovascular invasion (LVI) [Figure 2]a while 63.5% of cases showed axillary lymph node metastasis [Figure 2]b. It was found that LVI positive tumors showed increased number of axillary lymph node metastasis and this association was found to be statistically significant [Table 2]. There was also an association noted with increasing frequency of lymph node metastasis with increasing size and grade of the tumor [Table 3].
Figure 2: (a) Lymphovascular invasion – The tumor cells are seen invading a blood vessel. Also seen are RBCs within the vessel (H and E, ×40); (b) Axillary lymphnode metastasis – Section of a lymph node showing tumor deposit almost completely replacing the lymph node. Peripheral rim of normal lymphoid tissue is seen along with the capsule (H and E, ×40); (c) Tumor necrosis – Section shows IDC with wide areas of necrosis (H and E, ×100); (d) Stromal reaction - Section shows IDC with stroma showing extensive infiltration by lymphocytes (H and E, ×100)

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Table 2: Correlation of LVI with axillary lymph node involvement

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Table 3: Patient and tumor characteristics stratified by Axillary lymph node metastasis status

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There was tumor necrosis [Figure 2]c in 58.8% of cases and the incidence increased with increasing grades of tumor. It was observed that 80.6% of grade III tumors showed necrosis compared to 48.6% of grade II tumors, while none of the grade I tumors showed any necrosis.

Stromal reaction [Figure 2]d was observed in 52.9% of cases and in contrast to tumor necrosis, the stromal reaction decreased with increasing grade of the tumor.

As assessed by microscopic examination, the deep resected margin was seen to be involved in 8.2% of cases and 2.4% of cases showed involvement of the nipple and areola.

The results of MVD counts recorded independently by the two observers showed no significant interobserver variability, and the average MVD count increased with increasing grade of the tumor. Correlating the MVD counts with the other prognostic indicators, it was observed that the average MVD was 4.63/HPF in lymph node positive cases and 4.48/HPF in lymph node negative cases. In tumors associated with stromal reaction, the MVD was 4.49/HPF compared to 4.58/HPF noted in tumors without stromal reaction. In contrast to these findings of increased MVD associated with the poor prognostic indicators, there was a lower MVD count noted in necrosis positive cases (4.39/HPF) as compared to a higher MVD count noted in necrosis negative cases (4.72/HPF), though presence of necrosis is a indicator of poor prognosis.

This finding was further supported on comparing the MVD counts with the different grades of IDC, [Table 4] were it was observed that the average MVD counts in grade II and grade III tumors were higher in necrosis negative cases as compared to the necrosis positive cases.
Table 4: Comparison of MVD and other prognostic markers in the different grades of IDC

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Immunohistochemical status was determined in 50 cases of IDC to note the ER, PR, and HER-2/neu status. ER was positive [Figure 3]a in 12 cases, PR was positive [Figure 3]b in 15 cases, and HER-2/neu was positive [Figure 3]c in 27 cases.
Figure 3: (a) Estrogen receptor – Tumor cells showing nuclear positivity with estrogen receptor immunostain (ER, ×100); (b) Progesterone receptor – Tumor cells showing nuclear positivity with progesterone receptor immunostain (PR, ×100); (c) HER-2/neu – Tumor cells showing membrane positivity with HER-2/neuimmunostain (HER-2/neu, ×200)

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It was observed that the ER and PR positive tumors were smaller in size, of lower grade, and they did not show LVI more often, lymph node involvement or necrosis while HER-2/neu amplification was associated with larger tumor size, higher grade, and they more often showed LVI, lymph node involvement, and necrosis.

There were 15 triple negative tumors of which 86.66% were of T2 category, 40% showed LVI, 66.66% showed lymphnode metastasis, and 60% showed tumor necrosis.

Correlating the prognostic factors with the histological grade [Table 5], it has been observed that majority of the patients under the age of 40 (54.5%) had grade II tumors, while those over the age of 40 years (51.7%) had grade III tumors. The average size of grade I tumors was 3 cm while that of grade II AND III tumors were 4.12 cm and 4.60 cm, respectively.
Table 5: Comparison of histological grade with the other prognostic indicators in IDC cases

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None of the grade I tumors showed lymphovascular invasion while it was seen in 24.24% of grade II and 41.66% of grade III tumors. Similar to LVI, grade I tumors showed no lymph node involvement, while the average number of lymph nodes involved in grade II tumors was 3.03 and in grade III tumors, it was 4.69 lymph nodes. Majority of grade III tumors (80.55%) showed tumor necrosis in contrast to grade I tumors that showed no necrosis while 48.48% of grade II tumors showed necrosis. Similar to these findings the average MVD count increased with increasing grades of tumor but on comparing the MVD counts in cases with and without necrosis, the counts of both grade II and III cases were lower in necrosis positive cases (in comparison with cases without necrosis) though presence of necrosis is a indicator of poorer prognosis. In contrast to all these prognostic features which increased with increasing grades of tumor the stromal reaction decreased with increasing grade of tumor.

ER was positive in all cases of grade I, 34.61% of grade II, and 9.09% of grade III tumors. PR was positive in all cases of grade I, 38.46% of grade II tumors, and 13.63% of grade III tumors. HER-2/neu was seen to be amplified in none of grade I tumors, in 50% of grade II, and in 63.63% of grade III tumors. Thus, it is seen that ER and PR positivity decreased with increasing grades of the tumor while amplification of HER-2/neu increased with increasing grades of the tumor [Table 6].
Table 6: Comparison of hormonal status with histological grade of the tumor

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Correlating all the prognostic factors, it was observed that ER, PR negativity, and HER-2/neu amplification is associated with larger tumor size, higher grade, and they more often show LVI, lymph node metastasis and increasing necrosis.


 » Discussion Top


Breast cancer is a heterogenous disease with a wide range of clinical manifestations and histological types. As a result, a patient's individual prognosis at the time of diagnosis requires a detailed examination of as many clinical and pathological parameters as possible. Prognostic factors in breast cancer are indicators that reflect the individual characteristics of the tumor and the patient. Analysis and evaluation of these factors play a fundamental role in selection of the most effective cancer specific therapy with the least toxic effects and contribute to the prolonged survival and prediction of prognosis of patients diagnosed with breast cancer.[5] In the present study, various prognostic factors have been evaluated and correlated with their grades to note their significance.

The age range of the patients in the study was 28 to 82 years with a mean of 54.9 ± 13.5 years and a mean of 55 years. There were 15.4% of the patients who were below 40 years of age. These figures are comparable with the results of a study by Garicochea et al.[6]

Increasing incidence of lymph node metastasis and a decreased survival rate are associated with increasing tumor size.[7],[8] In the present study majority of T2 and T3 tumors (52.8% and 53.3%, respectively) were of grade III. The larger size and poor differentiation of tumors at the time of diagnosis could be due to lack of awareness and absence of mammographic screening protocols.

The study by Boiesen et al., concerning inter-observer variation of histological grade between seven pathology departments, have demonstrated a moderate degree of reproducibility, while still there are some concerns regarding the incorporation of grade into routine breast cancer staging system.[8],[9] Results of the present study have shown an association between the increasing grade of tumor and an increasing incidence of LVI, lymph node metastasis, tumor necrosis, and a decreasing incidence of stromal reaction.[10]

Angiogenesis begins at the earliest stage of an in-situ lesion and is required for further growth and metastasis of the tumor.[11] Weidner was the first to demonstrate that quantifying tumor neovascularization yields prognostically useful information in patients with invasive breast cancer.[12] Since then, several studies have reported conflicting results regarding the prognostic significance of MVD, with few studies finding an inverse relationship between survival and MVD, whereas others found no relation.[11] The present study indicates that MVD is a poor prognostic indicator, because though it shows a positive correlation with increasing tumor grade and lymphnode involvement, a higher MVD is noted in necrosis negative cases as compared to necrosis positive cases and thus showing a negative correlation with tumor necrosis. This reflects that MVD counts are dependent on the morphological architecture and it is seen to be affected in areas of necrosis.[13] These findings are comparable to the findings of our previous study with Shivakumar et.al., [Table 7] and more studies are indicated to confirm this.[14]
Table 7: Valves of MVD correlated with other prognostic markers and grades of IDC

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ER and PR expressions were associated with lower histological grade, smaller tumor size, fewer lymph node metastasis, and lower incidence of LVI and necrosis. These findings of ER expression are similar to those of Goyanes et al.,[5] PR had lost its dependent predictive value during the 1990s.[15] However, the existence of a number of ER negative patients who respond to hormone therapy has renewed interest in studying the response range to this type of therapy. PR has now regained importance and has been redefined as a predictive marker of ER activity, of growth factors, and also as a fundamental marker for indicating hormone therapy in breast cancer patients.[16]

HER-2/neu is a marker of poor prognosis, and it may be a predictive marker of response to certain types of chemotherapy.[17] The HER-2/neu over expression associated with higher histological grade, larger tumor size, lymphnode metastasis, absence of stromal reaction and presence of LVI and necrosis is similar to the findings of Ponzone et al.[15] An inverse correlation of ER, PR expression to that of HER-2/neu amplification has been noted similar to that of Lal et al.[18]

The triple negative cases as reported by Ovcaricek et al., showed association with larger tumor size, poor differentiation, and lymphnode metastasis. Patil et al., have noted that the triple negative tumors are associated with more aggressive clinico-pathological features and shorter relapse free survival.[19],[20]


 » Conclusion Top


The traditional morphological factors including the histological type, grade, tumor size, lymphovascular invasion, lymphnode status, necrosis, stromal reaction, and MVD are relatively simple to assess. Robust prognostic factors, the hormonal and genetic status not only have a prognostic value but are useful predictive markers for adjuvant chemotherapy.

In future, it is likely that a molecular classification of breast cancer will become a common requirement and treatment strategies will be based on specific genetic or biological events comparable with the current use of hormone therapy.


 » Acknowledgment Top


Dr. MuraliDhar, Assistant Professor, Department of Community Medicine. Dr. Manjunath Gubannana V., DCP, M.D., Professor and Head, Department of Pathology, JSS Medical College, JSS University, Mysore.

 
 » References Top

1.
Ahmad Z, Khurshid A, Qureshi A, Idress R, Asghar N, Kayani N. Breast carcinoma grading, estimation of tumor size, axillary lymph node status, staging, and Nottingham prognostic index scoring on mastectomy specimens. Indian J PatholMicrobiol 2009;52:477-81.  Back to cited text no. 1
    
2.
Lester SC. The Breast. In: Kumar V, Abbas AK, Fausto N, Aster JC, editors. Robbins and Cotran. Pathologic Basis of Disease. 8th ed. Philadelphia: Elsevier; 2010. p. 1065-95.  Back to cited text no. 2
    
3.
Ellis IO, Pinder SE, Lee AH. Tumors of the breast. In: Fletcher CD. Diagnostic Histopathology of Tumors. Vol. 1. 3rd ed. Philadelphia: Churchill Livingstone; 2007. p. 903-70.  Back to cited text no. 3
    
4.
Donegan WL. Tumor related prognostic factors for breast cancer. CA Cancer J Clin 1997;47:28-51.  Back to cited text no. 4
    
5.
Alvarez Goyanes RI, Escobar Pérez X, Camacho Rodríguez R, Orozco López M, Franco Odio S, Llanes Fernández L, et al. Hormone receptors and other prognostic factors in breast cancer in Cuba. MEDICC Rev 2010;12:36-40.  Back to cited text no. 5
    
6.
Garicochea B, Morelle A, Andrighetti AE, Cancella A, Bós A, Werutsky G. Age as a prognostic factor in early breast cancer. Rev Saude Publica 2009;43:311-7.  Back to cited text no. 6
    
7.
Fitzgibbons PL, Page DL, Weaver D, Thor AD, Allred DC, Clark GM, et al. Prognostic factors in breast cancer: College of American Pathologists Consensus Statement 1999. Arch Pathol Lab Med 2000;124:966-78.  Back to cited text no. 7
    
8.
Michaelson JS, Silverstein M, Sgroi D, Cheongsiatmoy JA, Taghian A, Powell S, et al. The effect of tumor size and lymph node status on breast carcinoma lethality. Cancer 2003;98:2133-43.  Back to cited text no. 8
    
9.
Boiesen P, Bendahl PO, Anagnostaki L, Domanski H, Holm E, Idvall I, et al. Histologic grading in breast cancer-Reproducibility between seven pathologic departments. South Sweden Breast Cancer Group ActaOncol 2000;39:41-5.  Back to cited text no. 9
    
10.
Rakha EA, El-Sayed ME, Lee AH, Elston CW, Grainge MJ, Hodi Z, et al. Prognostic significance of Nottingham histologic grade in invasive breast carcinoma. J ClinOncol 2008;26:3153- 8.  Back to cited text no. 10
    
11.
Kanjanapanjapol S, Wongwaisayawan S, Phuwapraisirisan S, Wilasrusmee C. Prognostic significance of microvessel density in breast cancer of Thai women. J Med Assoc Thai 2007;90:282-90.  Back to cited text no. 11
    
12.
Thorpe SM. Estrogen and progesterone receptor determinations in breast cancer. Technology, biology and clinical significance. Acta Oncol 1988;27:1-19.  Back to cited text no. 12
    
13.
Hasebe T, Tamura N, Iwasaki M, Okada N, Akashi-Tanaka S, Hojo T, et al. Grading system for lymph vessel tumor emboli: Significant outcome predictor for patients with invasive ductal carcinoma of the breast who received neoadjuvant therapy. Mod Pathol 2010;23:581-92.  Back to cited text no. 13
    
14.
Shivakumar S, Prabhakar BT, Jayashree K, Rajan MG, Salimath BP. Evaluation of serum vascular endothelial growth factor (VEGF) and microvessel density (MVD) as prognostic indicators in carcinoma breast.J Cancer Res Clin Oncol 2008;135:627-36.  Back to cited text no. 14
    
15.
Bezwoda WR, Esser JD, Dansey R, Kessel I, Lange M. The value of estrogen and progesterone receptor determinations in advanced breast cancer. Estrogen receptor level but not progesterone receptor level correlates with response to tamoxifen. Cancer 1991;68:867-72.  Back to cited text no. 15
    
16.
Ponzone R, Montemurro F, Maggiorotto F, Robba C, Gregori D, Jacomuzzi ME, et al. Clinical outcome of adjuvant endocrine treatment according to PR and HER-2 status in early breast cancer. Ann Oncol 2006;17:1631- 6.  Back to cited text no. 16
    
17.
Mohsin SK. HER2 testing: State of the laboratories. Arch Pathol Lab Med 2010;134:660-2.  Back to cited text no. 17
[PUBMED]    
18.
Lal P, Tan LK, Chen B. Correlation of HER-2 status with estrogen and progesterone receptors and histologic features in 3,655 invasive breast carcinomas. Am J ClinPathol 2005;123:541-6.  Back to cited text no. 18
    
19.
Ovcaricek T, Frkovic SG, Matos E, Mozina B, Borstnar S. Triple negative breast cancer-prognostic factors and survival. Radiol Oncol 2011;45:46-52.  Back to cited text no. 19
    
20.
Patil VW, Singhai R, Patil AV, Gurav PD. Triple-negative (ER, PgR, HER-2/neu) breast cancer in Indian women. Breast Cancer: Targets and Therapy 2011;3:9-19.  Back to cited text no. 20
    


    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7]

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