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ORIGINAL ARTICLE  
Year : 2018  |  Volume : 61  |  Issue : 3  |  Page : 330-333
Detection of micrometastasis in axillary lymph nodes of breast carcinoma patients and its association with clinical outcome


1 Department of Pathology, Amrita Institute of Medical Sciences, Kochi, Kerala, India
2 Regional Public Health Lab, Kochi, Kerala, India
3 Department of Breast and Gynaecological Oncology, Amrita Institute of Medical Sciences, Kochi, Kerala, India

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Date of Web Publication13-Jul-2018
 

   Abstract 


Context: There is heterogeneity in the clinical behavior of breast carcinoma patients with node negativity. Studies have analyzed different factors influencing the outcome in such patients. It is suggested that the presence of nodal micrometastasis can act as a tool in predicting the aggressiveness of these tumors. Aims: The aim of this study is to assess the yield of micrometastasis/isolated tumor cell (ITC) by ultrastaging the morphologically negative axillary nodes and staining them with immunohistochemistry for epithelial membrane antigen. The association of such metastasis with the clinical outcome is determined. Settings and Design: This was a retrospective analytical study. One hundred cases of node-negative breast carcinoma patients who underwent surgery along with axillary lymph node dissection were selected. Materials and Methods: The largest node from the axillary dissection was selected and subjected to ultrastaging and immunohistochemical staining (as sentinel node dissection was not a routine practice at that time), to look for occult metastasis in the form of micrometastasis or ITCs. Statistical Analysis: Occurrence of events in the form of recurrence or death was noted. Association of the parameters was analyzed using Fisher's exact test. Results: Among the 100 cases, 79 patients were followed up for a minimum period of 5 years. Two cases had micromets in one node each. These two patients were among the eight, who developed events subsequently (25%). Hence, a statistically significant association was found between the presence of micromets with events. Conclusions: There is a statistically significant association between the presence of micromets and disease recurrence. Hence, we suggest that ultrastaging of the negative axillary node (now sentinel node, as it is being routinely done) might prove effective in predicting the events/prognosis in clinically and morphologically node-negative breast carcinoma patients

Keywords: Micrometastasis, node-negative breast carcinoma, ultrastaging

How to cite this article:
Nair IR, Mathew AJ, Kottarathil VD. Detection of micrometastasis in axillary lymph nodes of breast carcinoma patients and its association with clinical outcome. Indian J Pathol Microbiol 2018;61:330-3

How to cite this URL:
Nair IR, Mathew AJ, Kottarathil VD. Detection of micrometastasis in axillary lymph nodes of breast carcinoma patients and its association with clinical outcome. Indian J Pathol Microbiol [serial online] 2018 [cited 2020 Jun 3];61:330-3. Available from: http://www.ijpmonline.org/text.asp?2018/61/3/330/236627





   Introduction Top


Breast carcinoma is the most common cancer affecting women (23%) and carries the second highest mortality rate exceeded only by lung cancer.[1],[2],[3] It is known that the malignant cells disseminating from the tumor are first harbored in the regional lymph nodes and will remain quiescent for variable time before entering the systemic circulation. Hence, sentinel node biopsy is sensitive and specific for predicting metastasis.[4] Although axillary lymph node metastasis remains the single most important prognostic factor in breast carcinoma patients, the fact that around 25% of patients without axillary node metastasis subsequently develop distant metastasis has prompted the search for other prognostic factors.[5] One approach has been to investigate the negative nodes thoroughly in an attempt to find metastasis missed by conventional light microscopic assessment.

Metastatic deposits of size 0.2–2 mm are considered micrometastasis (micromets). Single cells/deposit <0.2 mm are called isolated tumor cells (ITC). While the significance of macrometastasis is well established in the staging of tumor, it still remains unclear whether detection of micromets or ITC in the nodes influences the prognosis and long-term survival of breast carcinoma patients. These types of metastasis are best detected by ultrastaging and immunohistochemistry (IHC) using epithelial cell markers such as epithelial membrane antigen (EMA) and cytokeratin CK (AE1/AE3).

Aims

This study is done to assess the yield of micrometastasis/ITC by taking multiple level sections from the morphologically negative axillary nodes (ultrastaging) and staining them with IHC for EMA.

The association of such metastasis with the clinical outcome of these patients is also determined.


   Materials and Methods Top


This is a retrospective analytical study. One hundred patients with breast carcinoma who attended the surgical oncology department, during the period January 2004 to December 2006, and who were negative for nodal metastasis on clinical and routine morphological examination were selected. All these patients had undergone wide local excision/mastectomy along with axillary lymph node dissection (ALND) and were found to be negative for nodal metastasis on routine hematoxylin and eosin staining (p N0). Sentinel node biopsy had not become a routine practice at that time, and hence, all the cases had axillary clearance. As per the standard grossing practice, the axillary fat was dissected thoroughly to identify the nodes and the remaining fat was put in chloroform solution for better identification of the small nodes. Any node <5 mm was embedded fully. Nodes >5 mm were bisected and fully embedded.

Subjecting all the nodes to ultrastaging is time-consuming and costly, and hence, all the lymph node sections were reviewed for morphological identification of metastasis and confirmed as negative. The largest lymph node was then selected and nine serial step sections were taken from each paraffin block. Three sections, each of 3 micron thickness, were taken and mounted on poly-l-lysine-coated slides. The 1st, 4th, and 7th sections were taken on the first slide and labeled, the 2nd, 5th, and 8th sections on the second slide, and the 3rd, 6th, and 9th sections on the third slide. Routine hematoxylin and eosin staining was performed on the first slide. Sections were examined, and if any suspicious cells are found, adjacent sections (in the next unstained slide) were subjected to IHC staining. If no suspicious cells were seen, sections in the second and third were subjected to IHC staining. IHC was done using monoclonal antibodies for EMA (catalog number – MC 5 prediluted, Dako, Germany).

The clinical details and follow-up were obtained from the electronic medical records. Clinical outcome was found out in all the patients. The association of micromets with the occurrence of events, that is, disease recurrence/metastasis and/or death due to this disease was determined.


   Results Top


Among the 100 cases of breast carcinoma cases selected, 88 were infiltrating duct carcinoma, 7 infiltrating lobular carcinomas, and 5 other subtypes (two invasive papillary, two tubulolobular, and one medullary). The number of axillary nodes dissected in each case varied from 3 to 30 (with a mean of 16).

Among the nodes subjected to IHC staining, EMA-positive tumor cells were seen in one lymph node each from two different cases. One showed micrometastasis of 1.7 mm size. Small cluster of cells was seen [Figure 1]. Other node showed dyscohesive cluster of tumor cells, measuring 1.5 mm in maximum dimension. Macro metastasis or ITC was not detected. All other IHC-stained nodes were negative for metastasis.
Figure 1: Immunohistochemistry for epithelial membrane antigen shows micromets in the lymph node

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Ninety-six patients were followed up for a minimum period of 1 year, that is, 17 only for a year, 38 for 5 years, and rest 41 for 10 years till December 2016. Hence, the minimum period of 5-year follow-up was available for 79 patients. During this period, eight patients had developed events, in the form of local recurrence in three and distant metastasis in five. Three of the five patients with metastasis succumbed to the illness. Rest five (2 with mets and 3 with recurrence) were alive at the end of 5 years. Five others died of unrelated causes (not included in the events), including second malignancies in three (chronic lymphocytic leukemia, stomach carcinoma, and cervical carcinoma ). Of the eight cases with events, two were the ones identified to have micromets by ultra staging and IHC staining. These patients developed bone metastasis by 3 years and 5 years, respectively. The association of micromets with events was calculated using the Fisher's exact test and was found to be statistically highly significant (P = 0.006). The sensitivity, specificity, and positive and negative predictive values of micromets on clinical outcome were also calculated. The specificity and positive predictive value were found to be 100%. Sensitivity was found to be 25% while negative predictive value was 93%.

On stratifying the cases according to the disease stage [Table 1], it was found that most (90%) of the patients were of T2N0 stage. Nearly 4% were T1 or TX (when surgery was done outside and only tissue blocks were reviewed). The number of T3/T4 cases were less (5 in T3 and 1 in T4) as high-stage diseases with node negativity are rare to occur. Maximum number of events occurred in the T2 stage patients followed by T3 [Table 1]. No events were noted in T1 and T4 stages. We could not find any statistically significant difference in the occurrence of events between T2 stage and others (Chi-square test, P = 0.334). This may be due to the less number of cases in non-T2 stages. Hence, stage was not found to be an independent factor in predicting the occurrence of events.
Table 1: Number of events and micromets in cases in each stage

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At 5 years of follow-up, all T1 patients were found to be alive (100%) and 95.6% of T2 and all of T3 and T4 patients were alive. Similarly, 89.8% of T2 stage patients were found to be disease free at 5 years. Overall survival at 5 years was 96.2%.


   Discussion Top


Although it is well proven that the single most important prognostic factor in breast carcinoma patients is axillary nodal status, there is marked heterogeneity in clinical behavior among the women with lymph node mets. This has led to the subcategorization of mets into macromets, micromets, and ITCs by Huvos et al. in 1971 and was later incorporated into the AJCC TNM in 1984.[6] Since then, several studies have assessed the effect of micromets and ITC on clinical outcome, giving variable results. Most of these studies have shown the detection of occult metastasis in 9%–30% of cases, by IHC.[7] Byrne et al. studied all the dissected nodes from each case by step sectioning and IHC (EMA).[8] G Cserni also studied occult metastasis in all the lymph nodes in their cases, using multiple antibodies such as EMA, CK, and MUC-1.[9] We could detect micromets in only 2% of the cases. This may be attributed to the limited number of nodes and sections subjected to ultra staging and IHC staining in our series, which must also have led to the low sensitivity (25%) for predicting the worse outcome in cases. IBCSG 23-01 trial, carried out from 2001 to 2010 at the European Institute of Oncology (IEO), included patients with T <5 cm and absent clinically suspicious axillary lymph nodes.[4] The patients underwent a 5-year follow-up, resulting in a low (<1%) final local recurrence rate in group without ALND, and finally, no statistically significant difference in survival between the two groups was observed. Although these results were promising, in the group treated with ALND, an involvement of nonsentinel lymph node (SLN) in 13% of cases was found. The study was limited by the small number of patients due to selection criteria. The data obtained led to the St. Gallen Consensus Conference of 2011 to amend the recommendations in this direction, advising people to avoid ALND for patients with SLN micrometastases.[10]

The ASCO study in 2009 analyzed 1000 cases with ITC/micromets and found that the chance of recurrence in patients with micromets is 5 times more than the node-negative patients if there is no further treatment given, in the form of ALND or radiotherapy. Later on, the clinical trial conducted in the National Cancer Institute (ACOSOG ZOO 10) in 2011 concluded that the patients with micromets in sentinel node can avoid ALND without reducing the likelihood of survival.[11] However, the study published by Rosen et al. found that although at a follow-up of 6 years, there is no difference in survival of cases with micromets as compared with the node-negative patients, at the time of 12 years, there was a statistically significant decrease in survival.[12] We had a 5-year follow-up which showed a significant association of micromets with events. There are more data from Milan, from a retrospective study carried out at the same institution (IEO). Three hundred and seventy-seven patients with micrometastases to SLN between 1999 and 2007 were treated for breast cancer without ALND for any reason.[9] The 5-year survival was 97.3%, the cumulative incidence of local recurrences was 1.6%, and it seems that the tumor size and histological grade may have an important role. The authors concluded that in accordance with results of this trial, it is reasonable to discuss with the patient the chance to refuse any additional treatments when micrometastases in SLN are found, particularly for patients with small tumor (<2 cm), low histological grade, for the low risk of local recurrence. On comparing our observations, the overall survival was similar, 96.2%, but we could not find any significant association of stage with the adverse events. An inadequate number of patients in higher stages (T3/T4), for comparison, may be the reason for this difference. However, the comparative reduction in survival among the T2 stage patients (95.6%) could be attributed to the fact that a maximum number of patients belonged to that group. A statistically significant association of events was demonstrable with the presence of micromets in the largest axillary node. This study was done on the axillary nodal dissection as sentinel node biopsy was not the standard practice during that period of time. Hence, we suggest that with the current practice of routine sentinel node examination, whenever it is found to be negative on routine morphological examination, ultrastaging of the sentinel node might prove effective in predicting events/prognosis in clinically and morphologically node-negative breast carcinoma patients.


   Conclusions Top


There is heterogeneity in the clinical behavior of breast carcinoma patients with node negativity. Many studies have shown a significant association of micromets with disease recurrence. Although we have done ultrastaging of the largest node taken from axillary nodal dissection and not the sentinel node, we could find a statistically significant association with the patient outcome. Hence, ultrastaging of the clinically and morphologically negative sentinel node might prove to be a better tool in predicting the clinical outcome of breast carcinoma patients.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

1.
American Cancer Society: Cancer Facts and Figures; 2017 CRA504 ([Meeting Abstracts]).  Back to cited text no. 1
    
2.
Parkin DM. Global epidemiological methods: Use of statistics to assess the global burden of breast cancer. Breast J 2006;12:1.  Back to cited text no. 2
    
3.
Breast Cancer Mortality Statistics. Cancer Research UK: Cancer Breast Mortality; 2014.  Back to cited text no. 3
    
4.
Galimberti V, Cole BF, Zurrida S, Viale G, Luini A, Veronesi P, et al. Axillary dissection versus no axillary dissection in patients with sentinel-node micrometastases (IBCSG 23-01): A phase 3 randomised controlled trial. Lancet Oncol 2013;14:297-305.  Back to cited text no. 4
    
5.
Park H, Chang SK, Kim JY, Lee BM, Shin HS. Risk factors for distant metastasis as a primary site of treatment failure in early-stage breast cancer. Chonnam Med J 2014;50:96-101.  Back to cited text no. 5
    
6.
Huvos AG, Hutter R, Berg JW. Significance of axillary macro metastasis and micro metastasis in mammary cancer. Ann Surg 1971;173:44-6.  Back to cited text no. 6
    
7.
Dowlatshahi K, Fan M, Snider HC, Habib FA. Lymph node micrometastases from breast carcinoma: Reviewing the dilemma. Cancer 1997;80:1188-97.  Back to cited text no. 7
    
8.
Byrne J, Waldron R, McAvinchey D, Dervan P. The use of monoclonal antibodies for the histopathological detection of mammary axillary micrometastases. Eur J Surg Oncol 1987;13:409-11.  Back to cited text no. 8
    
9.
Cserni G. Metastases in axillary sentinel lymph nodes in breast cancer as detected by intensive histopathological work up. J Clin Pathol 1999;52:922-4.  Back to cited text no. 9
    
10.
Gnant M, Harbeck N, Thomssen C. St. Gallen 2011: Summary of the consensus discussion. Breast Care (Basel) 2011;6:136-41.  Back to cited text no. 10
    
11.
Cote R, Giuliano AE, Hawes D, Ballman KV, Whitworth PW, Blumencranz PW. ACOSOG Z0010: A multi center prognostic study of sentinel node (SN) and bone marrow (BM) micro metastases in women with clinical T1/T2 N0 M0 breast cancer. J Clin Oncol 2010;28:CRA504 ([Meeting Abstracts]).  Back to cited text no. 11
    
12.
Rosen PP, Saigo PE, Braun DW, Weathers E, Fracchia AA, Kinne DW, et al. Axillary micro- and macrometastases in breast cancer: Prognostic significance of tumor size. Ann Surg 1981;194:585-91.  Back to cited text no. 12
    

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Correspondence Address:
Indu Ramachandran Nair
Department of Pathology, Amrita Institute of Medical Sciences, Ponekkara, Edapally, Kochi - 682 041, Kerala
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/IJPM.IJPM_741_17

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Ranjan Agrawal
Indian Journal of Pathology and Microbiology. 2018; 61(3): 309
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