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ORIGINAL ARTICLE  
Year : 2012  |  Volume : 55  |  Issue : 2  |  Page : 183-186
Comparing touch imprint cytology, frozen section analysis, and cytokeratin immunostaining for intraoperative evaluation of axillary sentinel lymph nodes in breast cancer


1 Department of Pathology, Shiraz University of Medical Sciences, Shiraz, Iran
2 Department of Surgery, Shiraz University of Medical Sciences, Shiraz, Iran

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Date of Web Publication3-Jul-2012
 

   Abstract 

Background: Sentinel lymph node (SLN) biopsy has been applied to the management of breast carcinoma inorder to decrease postoperative complication and morbidity. Touch imprint cytology (TIC), frozen section (FS), scrape cytology, or combination of these methods are used as intraoperative diagnostic methods. However, the sensitivity of these intraoperative modalities for detecting metastatic disease in SLNs is not equivalent to permanent histopathologic examination as a gold standard method. Objectives: The aim of this study was to review our department's results with SLN biopsy using touch imprint and frozen section for intraoperative diagnosis of breast cancer metastasis. Immunohistochemistry for cytokeratin was used on permanent sections. The sensitivities and specificities of TIC with those of FS analysis and IHC were also compared. Materials and Methods: A total of 100 consecutive SLN biopsies from 49 patients performed. The TIC and subsequently frozen were stained using hematoxylin and eosin. The cytological and frozen findings were compared and results were reported to the surgeon during operation. Final pathologic evaluation was performed on the formalin-fixed, paraffin-embedded tissue sections. Analysis of the permanent tissue included evaluation of three-step sections of the lymph node by H&E and immunohistochemical (IHC) staining. The sensitivities, specificities, positive and negative predictive values of TIC, FS and IHC for the detection of metastatic tumor in the SLNs were determined with the corresponding 95% confidence intervals (CIs). Results: One hundred SLNs were examined from 49 patients with invasive breast carcinoma with mean age of 45.29 ° 10.6 years. Intraoperative TIC and FS failed to show metastatic involvement in 10 examined lymph nodes from three patients. No false positive results for TIC and FS was identified. The sensitivity of TIC compared with the final histopathological result, considered the gold standard, was 90% (CI, 68.49-98.81%). Similarly, the sensitivities of frozen sections and permanent were the same respectively. The specificities of TIC, FS, and permanent were 100% (CI, 94.95-100.00). The sensitivity of touch imprint cytology compared with the final histopathological result, considered the gold standard, was 90% (CI, 68.49--98.81%). Similarly, the sensitivities of frozen sections and permanent were the same respectively. The specificities of TIC, FS, and permanent were 100% (CI, 94.95-100.00). Conclusions: Our experience with TI and FS for the intraoperative evaluation of SLNs is similar to the findings from previously reported studies. We detected the same sensitivities for these two methods; however lower sensitivity of TI in detecting metastasis with higher false-negative rate has been addressed in the published literature. The 90% sensitivity of TI and FS with permanent histopathologic examination as the gold standard falls within the range of reported sensitivities: 33-96% for TI and 44-100% for FS. However, variations in patient selection criteria, experience of the pathologist, skill of the technician submitting specimen for intraoperative evaluation, and tumor size are important variables that influence the results.

Keywords: Breast, cancer, sentinel lymph node

How to cite this article:
Safai A, Razeghi A, Monabati A, Azarpira N, Talei A. Comparing touch imprint cytology, frozen section analysis, and cytokeratin immunostaining for intraoperative evaluation of axillary sentinel lymph nodes in breast cancer. Indian J Pathol Microbiol 2012;55:183-6

How to cite this URL:
Safai A, Razeghi A, Monabati A, Azarpira N, Talei A. Comparing touch imprint cytology, frozen section analysis, and cytokeratin immunostaining for intraoperative evaluation of axillary sentinel lymph nodes in breast cancer. Indian J Pathol Microbiol [serial online] 2012 [cited 2020 Apr 2];55:183-6. Available from: http://www.ijpmonline.org/text.asp?2012/55/2/183/97859



   Introduction Top


The sentinel lymph node (SLN) is defined as the first lymph node to drain the area under investigation. SLN biopsy has been applied to the management of several malignancies, including breast carcinoma and melanoma and it has had a good impact on decreasing postoperative complication and morbidity. In the past, widespread axillary dissection in breast cancer carried the risk of decreased shoulder range of motion and lymphedema with impact on patients' quality of life. [1],[2],[3]

Intraoperative evaluation consisted of touch imprint cytology (TIC), frozen section (FS) analysis, scrape cytology or combination of these methods with own advantages and disadvantages. [4],[5] TIC is the most common method of analysis of the SLN due to its rapid results, lack of artifact imposed by FS, and decreased cost. [1],[2],[3],[4],[5]

However, the sensitivities of these intraoperative methods for detecting metastatic disease in SLNs is not fully equivalent to permanent section examination as a gold standard method. Therefore, these tests can result in false-negative report of lymph node involvement by tumoral cells. [4],[5],[6] Other available techniques, such as rapid cytokeratin immunostaining and molecular methods, were reported to improve the sensitivity of intraoperative evaluation SLNs in breast cancer. [7]

The purpose of this study was to review our department's results with SLN biopsy using touch imprint and frozen section for intraoperative diagnosis of metastasis. Immunohistochemistry (IHC) for cytokeratin was used as another test to find metastasis on permanent section. We also compared the sensitivities and specificities of TIC with those of FS analysis and IHC for intraoperative evaluation of SLNs in breast cancer.


   Materials and Methods Top


A total of 100 consecutive SLN biopsies from 49 patients performed at our surgical center from January 1, 2005, through April 1, 2009. The study was approved by ethic committee of the University of Medical Sciences and Informed consent was obtained from all participants. Preoperative evaluation of axillary lymph node status was obtained by clinical examination and sonography.

SLNs received fresh in the FS suite and trimmed of excess adipose tissue. Sentinel nodes were bivalved along their long axis and imprinted directly on to microscopy slides. The imprints from each bivalved node were made on a separate microscope slide, fixed in 95% alcohol, and stained by the hematoxylin and eosin (H and E) method. Cases were subsequently frozen section and stained, also using hematoxylin and eosin. The cytological and frozen section findings were compared and results were reported to the surgeon during operation. Final pathologic evaluation was performed on the formalin-fixed, paraffin-embedded tissue sections of the lymph nodes. Analysis of the permanent section included evaluation of multiple sections of the lymph node by hematoxylin and eosin (H and E) with immunohistochemical (IHC) staining for cytokeratin AE1/AE3 (pan-cytokeratin) (Dako, Denmark) with positive and negative controls. The IHC was performed on paraffin-embedded tissue. Results of the TIC were compared with the definitive postoperative histopathology results.

Statistical Analysis

The sensitivities, specificities, positive and negative predictive values of TIC, FS and IHC for the detection of metastatic tumor in the SLNs were determined with the corresponding 95% confidence intervals (CIs). In the calculations, the data obtained by permanent section evaluation by H and E and cytokeratin immunostaing were considered as the gold standard.


   Results Top


In this study, 100 SLNs were examined from 49 patients with invasive breast cancer (average node yield, 2 nodes per patient). Mean age of the patients was 45.29 ° 10.6 years (range, 302-60 years). The quality of the FS and TIC was satisfactory in all patients for histological and cytological interpretation. IHC revealed strong cytoplasmic and membranous staining of the metastatic tumor cells [Figure 1].
Figure 1: The IHC staining pattern of metastatic tumor cells (AE1 ⁄ AE3 immunolabeling, × 40)

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[Table 1] summarizes the detection of the metastatic tumor in sentinel nodes by different methods. [Figure 2] illustrates metastatic tumor cells in cytology of the SLN. Intraoperative TIC and FS failed to show metastatic involvement in 10 examined lymph nodes from three patients. All false negative results of touch imprint cytology and frozen sections involved samples containing either isolated tumor cells or small foci of metastases.
Figure 2: (a and b) Touch imprint of metastatic ductal carcinoma in the sentinel node (H and E, × 40)

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Table 1: Cytological and pathologic findings of sentinel lymph nodes in breast carcinoma

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No false positive result in touch imprint cytology and frozen section was identified.

Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) have been calculated separately based on the number of the sentinel lymph nodes [Table 2]. The sensitivity of touch imprint cytology was 90% (CI, 68.49-98.81%), when compared with the final histopathological result as a gold standard method. Similarly, the sensitivity of frozen and permanent sections was the same respectively. The specificity of TIC, FS, and permanent were 100% (CI, 94.95-100.00). The sensitivity of touch imprint cytology was 90% (CI, 68.49-98.81%). Similarly, the sensitivity of frozen and permanent sections was the same respectively. The specificity of TIC, FS, and permanent sections were 100% (CI, 94.95-100.00).
Table 2: Comparison of different methods with the final pathological result in the evaluation of sentinel nodes

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   Discussion Top


Although SLN biopsy has provided decreased patient morbidity with breast carcinoma, but recurrent disease still occurs. Therefore, researches try to find the best evaluative approach in the management of this disease. [1],[2],[5]

Tew et al. [2] calculated the sensitivity of four different studies that compared TI with FS. They found sensitivity of 62% and 76% for TI and FS respectively. They reported a sensitivity range of 44--100% and a specificity of 100%, including all analytical techniques. The sensitivity and specificity of TIC were paralleled with FS analysis, without a statistically significant difference. [2]

Our experience with TI and FS for the intraoperative evaluation of SLNs is similar to the findings from previously reported studies. We detected the same sensitivities for these two methods; however lower sensitivity of TI in detecting metastasis with higher false-negative rate has been addressed in the published literature. [2],[7]

In the literature, the sensitivity of 33--96% for TI and 44--100% for FS was reported, which was similar to our findings. [2],[3],[4],[5],[6]

Variations in patient selection criteria as well as gross and permanent section account for the wide range of results from different studies. TI cytology has been compared in numerous studies against FS, and no statistically significant difference has been found between these two methods. [2],[6]

On comparison of TI and FS, although we observed that FS demonstrated higher sensitivity for detecting metastasis in general, the difference was not statistically significant. The lowered sensitivity of TI usually is caused by sampling, in that the metastasis is uncovered after serial cutting of the tissue block. The majority of the previous studies that directly compared TI with FS analysis in SLNs arrived at similar conclusions. [8] To our knowledge, to date, only Motomura et al. [8] have reported that TI cytology was better than FS. Our findings were similar to Brogie et al. [9] which demonstrated that TI cytology and FS were comparable for detecting metastases in SLNs. The experience of the pathologist, skill of the technician submitting specimen for intraoperative evaluation, and tumor size are important variables that influence the results. [2] Sensitivity for SLN biopsy increased as the tumor size increased and decreased as the proportion of micrometastasis increased. [2]

Krishnamurthy et al. [6] investigated the intraoperative processing of SLNs using a rapid pan-cytokeratin immunohistochemical stain. The procedure required an average of 25 minutes (range 8-25 minutes). They observed sensitivity of 80% in comparison with 75% for FS and 45% for TIC alone. They reported that rapid cytokeratin immunohistochemistry (RCI) were better for detection of micrometastasis, as well as the ability to accurately measure the size of the metastasis. [6] Others have used cytokeratin immunohistochemistry as an intraoperative evaluation method and reported that it was similar to FS analysis for detecting macrometastasis. [7] However, it was slightly better than FS for detecting small-sized metastasis and also lobular carcinomas. [6],[7] Aihara et al. [10] reported a slight improvement in sensitivity with the combination of TI cytology and RCI (from 83% to 85%).

IHC was useful in avoiding false-negative results in patients with metastatic lobular carcinoma, as demonstrated by Weinberg et al. [11] The utility of RCI in the evaluation of TI cytology in patients with invasive lobular carcinoma was tested and the sensitivity increased from 41.9% to 54.8% in the detection of metastases. [11]

Molecular techniques are other assays for the intraoperative analysis of SLN biopsies. Blumencranz et al. [12] developed a molecular assay for detection of axillary lymph node metastasis and found good results for macrometastases. They used the polymerase chain reaction amplification of extracted DNA with primers for cytokeratin. [12],[13],[14] Martin--Martinez et al. [13] compared the Gene Search BLN assay with intraoperative histologic evaluation as gold standard method. Results were similar to the histologic findings. Tsujimoto et al. [14] also found similar findings. Unlike the previous studies, this study also reported potential for detection of micrometastatic disease.

Molecular tests on automated platforms, such as the Gene Search BLN assay (Veridex, LLC, Warren, NJ) and the 1-step nucleic acid amplification assay (Sysmex, Japan), are available for intraoperative use. [15] However, these methods are impractical at present for practicing in the intraoperative setting due to the cost and time limitation and needs trained staff to set up such assays. On the other hand, few clinical trials showed that SLN occult metastases detected by serial step sections at 85 μm intervals did not have significant prognostic implications. [16] Others found that completion axillary dissection was not necessary in patients with positive SLNs. Therefore due to this controversy, future studies for intraoperative evaluation of SLNs are recommended.

 
   References Top

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2.Tew K, Irwig L, Matthews A, Crowe P, Macaskill P. Meta-analysis of sentinel node imprint cytology in breast cancer. Br J Surg 2005;92:1068-80.  Back to cited text no. 2
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3.Kootstra JJ, Hoekstra-Weebers JE, Rietman JS, de Vries J, Baas PC, Geertzen JH, et al. A longitudinal comparison of arm morbidity in stage I-II breast cancer patients treated with sentinel lymph node biopsy, sentinel lymph node biopsy followed by completion lymph node dissection, or axillary lymph node dissection. Ann Surg Oncol 2010;17:2384-94.  Back to cited text no. 3
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4.Creager AJ, Geisinger KR, Perrier ND, Shen P, Shaw JA, Young PR, et al. Intraoperative imprint cytologic evaluation of sentinel lymph nodes for lobular carcinoma of the breast. Ann Surg 2004;239:61-6.  Back to cited text no. 4
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5.Van de Vrande S, Meijer J, Rijnders A, Klinkenbijl JH. The value of intraoperative frozen section examination of sentinel lymph nodes in breast cancer. Eur J Surg Oncol 2009;35:276-80.  Back to cited text no. 5
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6.Krishnamurthy S, Meric-Bernstam F, Lucci A, Hwang RF, Kuerer HM, Babiera G, et al. A prospective study comparing touch imprint cytology, frozen section analysis, and rapid cytokeratin immunostain for intraoperative evaluation of axillary sentinel lymph nodes in breast cancer. Cancer 2009;115:1555-62.  Back to cited text no. 6
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7.Pargaonkar AS, Beissner RS, Snyder S, Speights VO Jr. Evaluation of immunohistochemistry and multiple-level sectioning in sentinel lymph nodes from patients with breast cancer. Arch Pathol Lab Med 2003;127:701-5.  Back to cited text no. 7
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8.Motomura K, Inaji H, Komoike Y, Kasugai T, Nagumo S, Noguchi S, et al. Intraoperative sentinel lymph node examination by imprint cytology and frozen sectioning during breast surgery. Br J Surg 2003;87:597-601.  Back to cited text no. 8
    
9.Brogi E, Torres-Matundan E, Tan LK, Cody HS 3 rd . The results of frozen section, touch preparation, and cytological smear are comparable for intraoperative examination of sentinel lymph nodes: A study in 133 breast cancer patients. Ann Surg Oncol 2005;12:173-80.  Back to cited text no. 9
    
10.Aihara T, Munakata S, Morino H, Takatsuka Y. Touch imprint cytology and immunohistochemistry for the assessment of sentinel lymph nodes in patients with breast cancer. Eur J Surg Oncol 2003;29:845-8.  Back to cited text no. 10
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11.Weinberg ES, Dickson D, White L, Ahmad N, Patel J, Hakam A, et al. Cytokeratin staining for intraoperative evaluation of sentinel lymph nodes in patients with invasive lobular carcinoma. Am J Surg 2004;188:419-22.  Back to cited text no. 11
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12.Blumencranz P, Whitworth PW, Deck K, Rosenberg A, Reintgen D, Beitsch P, et al. Sentinel node staging for breast cancer: Intraoperative molecular pathology overcomes conventional histologic sampling errors. Am J Surg 2007;194:426-32.  Back to cited text no. 12
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13.Martin-Martinez MD, Veys I, Majjaj S, Lespagnard L, Schobbens JC, Rouas G, et al. Clinical validation of a molecular assay for intra-operative detection of metastases in breast sentinel lymph nodes. Eur J Surg Oncol 2009;35:387-92.  Back to cited text no. 13
    
14.Tsujimoto M, Nakabayashi K, Yoshidome K, Kaneko T, Iwase T, Akiyama F, et al. One-step nucleic acid amplification for intraoperative detection of lymph node metastasis in breast cancer patients. Clin Cancer Res 2007;13:4807-16.  Back to cited text no. 14
    
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16.Takeshita T, Tsuda H, Moriya T, Yamasaki T, Asakawa H, Ueda S, et al. Clinical implications of occult metastases and isolated tumor cells in sentinel and non-sentinel lymph nodes in early breast cancer patients: Serial step section analysis with long-term follow-up. Ann Surg Oncol 2012;19:1160-6.  Back to cited text no. 16
    

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Correspondence Address:
Negar Azarpira
Organ Transplant Research Center, Department of Pathology, Nemazi Hospital, Shiraz University of Medical Sciences, Shiraz
Iran
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0377-4929.97859

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