ORIGINAL RESEARCH. International Journal of Surgery

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1 International Journal of Surgery 11 (2013) 253e258 Contents lists available at SciVerse ScienceDirect International Journal of Surgery journal homepage: Original research Intraoperative frozen section analysis of sentinel lymph nodes in breast carcinoma patients in a tertiary hospital in Pakistan Ambreen Moatasim *, Shafaq Mujtaba, Naveen Faridi Liaquat National Hospital, Karachi, Pakistan article info abstract Article history: Received 11 April 2012 Received in revised form 14 January 2013 Accepted 17 January 2013 Available online 23 January 2013 Keywords: Frozen section Sentinel lymph node Paraffin section Breast carcinoma Intraoperative evaluation of sentinel lymph nodes has become routine in many units that manage early breast carcinoma. The procedure is associated with minimal morbidity and can be cost effective, avoiding re operation and reducing hospital stay as an axillary clearance can be performed under the same anesthetic after a positive intraoperative diagnosis without awaiting conventional paraffin histology. In negative cases extensive axillary lymph node dissection and its associated side effects can be avoided altogether. With careful patient selection, the expertise of surgeons and pathologists can increase the sensitivity and specificity of the technique with a reduction in false negatives. A number of international studies have established the usefulness of intraoperative sentinel lymph node evaluation. However, no local study has assessed the accuracy of frozen section in evaluating sentinel lymph node biopsy. The purpose of our study was to compare the two techniques (frozen section versus conventional paraffin histology) of the sentinel lymph node examination and to present our local data highlighting its usefulness and pitfalls, comparing the results with those in published studies. From the results obtained, we strongly recommend intraoperative assessment of the sentinel lymph nodes in breast carcinoma patients; frozen section microscopy can be a reliable and accurate technique in the hands of an experienced histopathologist. Ó 2013 Surgical Associates Ltd. Published by Elsevier Ltd. All rights reserved. 1. Introduction Breast carcinoma is the second leading cause of cancer e related deaths among women worldwide 1 and the presence of axillary lymph node metastases has been regarded as a predictor of poorer survival. 2 Traditionally, axillary lymph node status has been determined by axillary lymph node dissection (ALND) which carries a recognized morbidity including the risk of paresthesiae or pain, hematoma, seroma, restricted shoulder movement and lymphedema. The only patients who can derive benefit from axillary lymph node dissection are those with positive nodes, accounting for 40% or fewer of all patients who undergo ALND. 3e5 If the sentinel node is free of tumor on frozen section, ALND and its associated morbidity can be avoided unless a metastasis or micrometastasis is evident on subsequent paraffin histology. Thirty two published studies 6 of the sentinel lymph node biopsy with back up axillary dissection have indicated that the sentinel lymph node can detect axillary metastases in 93% of node positive * Corresponding author. addresses: ambreen.moatasim@gmail.com (A. Moatasim), dr.shafaq@ hotmail.com (S. Mujtaba), naveenfaridi@hotmail.com (N. Faridi). patients, so many hospitals now offer intraoperative evaluation of sentinel lymph node (SLN) biopsy with no further axillary surgery needed in node negative cases. Having said this, frozen section assessment of the sentinel lymph nodes has been considered as time consuming, costly and subject to false negative results, with a reported sensitivity varying from 60 to 75%. 7e10 We report the experience of one breast unit with this technique and discuss the benefits and pitfalls. Intraoperative sentinel lymph node evaluation is being performed in limited centres in Pakistan, restricted by cost and limited experience amongst surgeons and histopathologists not comfortable and/ or adequately trained in this technique. Liaquat National Hospital Karachi is one of the few hospitals in Pakistan where this technique is routinely performed for managing breast carcinoma patients. In this study we have compared the two techniques, intraoperative frozen section and permanent paraffin sections, for evaluating sentinel lymph nodes in breast carcinoma patients. 2. Materials and methods This observational study was conducted from 2005 to 2010, in the Histopathology Department at Liaquat National Hospital, Karachi in collaboration with the Surgical Unit. All cases of breast carcinoma having clinically negative axillary lymph /$ e see front matter Ó 2013 Surgical Associates Ltd. Published by Elsevier Ltd. All rights reserved.

2 254 A. Moatasim et al. / International Journal of Surgery 11 (2013) 253e258 nodes were selected. Also included were post neoadjuvant cases with negative nodes. Neoadjuvant therapy was given to patients in whom the tumor was large, adherent to skeletal muscle or surgically unresectable. The neoadjuvant therapy consisted of adriamycin and cyclophosphamide for the first three months and docetaxel for the last three months with or without herceptin depending on Her-2- neu status. Cases with ALN involvement, proven either on histology or cytology prior to the main surgery were excluded from the study. Preoperative ultrasound assessment of axillary lymph nodes was not carried out in any of the cases. All breast tumors were preoperatively proven as positive for carcinoma on fine needle aspiration cytology (followed by confirmation on frozen section preceding any radical surgery) or incisional biopsy. Consent for surgery was taken by the department of surgery. The selected cases underwent lymphoscintigraphic mapping approximately 6 h prior to surgery. Technetium 99 labelled isotope was injected subepidermally in the peritumoral area in a dose of 37 mbq/4 injections of 9.2 mbq (volume ¼ 1 ml/4 injections of 0.25 ml). Sentinel lymph node biopsy was performed before removal of the primary tumor. The intraoperative frozen section assessment of the sentinel lymph node(s) was performed in the histopathology department with subsequent confirmation on paraffin sections. An operating room (OR) nurse accompanied the sentinel node specimen along with a request form. The histopathology staff used protective gloves, masks and disposable aprons while handling radioactive tissue. The time of receipt of lymph node(s) was recorded on the request form. After initial measurements and recording of the number of lymph nodes received, excess fat from each node was trimmed off. A small lymph node (¼ or <1 cm) was bisected and entirely submitted in one cassette. Lymph nodes >1 cm were serially sliced at 2 mm intervals perpendicular to the long axis and submitted entirely (not more than two slices in one cassette) to decrease the number of false negatives. A large lymph node was submitted in multiple cassettes. The specimen was then immediately frozen and thin sections were cut on a cryostat machine (Fig. 1). Sections were stained with Hematoxylin & Eosin. If on microscopy the first level of a lymph node was negative, a minimum of 3 additional levels of each section were examined. Any ambiguous section showing freezing and cutting artifacts was rejected and a new section was requested. A negative diagnosis was only given after all levels of submitted sections were examined carefully. In the case of a positive node an attempt was made to classify it as a micro or macrometastasis (the latter defined as a tumor deposit of more than 2 mm in size, a micrometastasis as a tumor deposit of 0.2e2 mm in size, and isolated tumor cells as single or clusters of tumor cells of less than 0.2 mm in aggregate size). Once a node was found to be positive, the examination of other lymph nodes being processed was halted. A positive or negative result was promptly communicated by telephone by the histopathologist to the operating surgeon who modified the surgery accordingly. This intraoperative assessment was undertaken by a single consultant histopathologist in rotation. In doubtful cases (of micrometastasis or single tumor cells especially) a second opinion by another colleague was sought. The tissue was then processed routinely and paraffin sections were examined to confirm the frozen section diagnosis. Where there was a discrepancy between the results of frozen and paraffin sections, the frozen section slides were re examined preferably by the primary reporting pathologist and re-checked by another consultant histopathologist. Immunohistochemistry, using CKAE1/AE3, was also available (Fig. 2). Before the final report was issued, the operating surgeon was informed of the change in status. In each case, patient and tumor demography were recorded. Statistical analysis was done by calculating sensitivity, specificity, accuracy, and positive and negative predictive values. 3. Results From 2005 to 2010, 100 patients meeting the selection criteria underwent intraoperative assessment of sentinel lymph nodes. All patients were female. The mean age of the patients was years with a standard deviation of 11.1 and median of 50 years. As the OR was on the same floor as the histopathology department, minimal time was wasted on sample transportation (<5 min). The operating surgeon while awaiting the sentinel biopsy report performed either a modified radical mastectomy or a breast conservation type complete local excision. The final specimens received in 100 cases were 67 mastectomies and 33 complete local excisions with cavity shavings sent separately. 7/100 surgeries followed post neoadjuvant therapy, with no residual tumor found after thorough sampling. In none of these had a hook wire been inserted preoperatively (Table 1). A total of 200 sentinel nodes were examined by frozen section, with a mean of 2 nodes per case. The number of nodes sent for intraoperative evaluation ranged from 1 to 6 (Table 2). The size of lymph nodes ranged from 0.5 to 3 cm in maximum dimension with a median of 2 cm and mean of 1.8 cm (0.67 SD). In 32 cases a positive diagnosis on frozen section was given (total nodes positive n ¼ 45). Where an axillary dissection specimen was available (84/100), an attempt was made to assess the final nodal status in cases reported negative on frozen section: no further metastasis was detected in any of these. However, in 4 additional cases, Fig. 1. Serial slicing and processing of a sentinel lymph node for frozen section.

3 A. Moatasim et al. / International Journal of Surgery 11 (2013) 253e Table 2 Frequency of nodes received. Number of nodes received Total Number of cases Total number of examined nodes Fig. 2. Subcapsular micrometastasis, confirmed on immunohistochemistry using cytokeratin AE1/AE3 (H&EX10M). Inset showing infiltrating ductal carcinoma Grade 2 as seen on paraffin section of breast lump of same patient. a metastatic tumor was confirmed on paraffin sections of lymph nodes reported negative on frozen section (Table 5). In 03/04 false negative cases, the complete nodal capsule was not properly visualized even on multiple levels and micrometastases were therefore missed. In 01/04 case, due to infiltration of lymph node by adipose tissue causing improper cutting, a focus of metastatic tumor was missed on frozen section. Immunohistochemistry using CKAE1/AE3 was performed on paraffin section to confirm the diagnosis in one case as there was a disagreement between two histopathologists (Fig. 2). The size of nodal (sentinel) metastases ranged from 1 to 20 mm with a mean of 7 mm. No false positive case was seen in this study. Primary tumor size is known to be an important predictor of nodal metastasis, so an attempt was made to correlate the tumor size with the presence of metastasis. The tumor size ranged from 1.2 to 6.5 cm with a median of 2.6 and mean of 2.7 cm (1.3 SD). The primary tumor size in sentinel nodes harboring macrometastases ranged from 1.8 to 6.5 cm with a mean of 3.48 and median of 3.35 cm whereas in nodes with micrometastases, the tumor size ranged from 2.3 to 2.8 cm with mean and median of 2.52 and 2.5 cm respectively. Table 3 compares tumor size with frequency of micro and macrometastases in the sentinel nodes. Table 1 Patient and tumor demography. Age range (years) 28e78 Mean age (years) (Standard deviation 11.1) Median age (years) 50 Mean tumor size (cm) 2.7 (Standard deviation 1.3) Axillary sampling (number of cases) 84/100 Histologic diagnosis Frequency Percentage (%) Infiltrating ductal carcinoma Infiltrating lobular carcinoma Ductal carcinoma in situ (DCIS) DCIS with microinvasion Infiltrating papillary carcinoma Infiltrating tubular/cribriform carcinoma Metaplastic carcinoma No residual tumor (status post neoadjuvant therapy) Solid papillary carcinoma Mixed (ductal and lobular) Total The mean time taken to report sentinel node biopsy was min with a median of 30 min (range 15e50 min) which fits well with the average time taken to perform a surgery i.e. 45e 50 min. The increase in reporting time was directly related to the number of nodes received, especially in negative cases in which at least three levels were examined. The results of SLN biopsy were communicated directly via telephone by the histopathologist to the operating surgeon or her assistant, with no subsequent evidence of any miscommunication of frozen section reports. The sensitivity, specificity, accuracy, and positive and negative predictive values based on number of patients and number of nodes can be seen in Table Discussion The technique of sentinel lymph node assessment was first introduced by Giuliano 11 and has recently become a standard procedure in centres dealing with a large number of breast carcinoma patients. According to the recommendations of the American Society of Oncology 12 SLN biopsy is an appropriate alternative to routine staging axillary lymph node dissection (ALND) for patients with early stage breast cancer having clinically negative axillary nodes. Complete axillary lymph node dissection remains standard treatment for patients with axillary metastases identified on the SLN biopsy. In experienced hands, properly identified patients with a negative result on SLN biopsy are not likely to require complete ALND. SLN biopsy is associated with less morbidity than ALND, 13 but the relative effects of these two approaches on tumor recurrence or patient survival are unknown. Like surgeons, pathologists evaluating the SLN specimens should be experienced, and should also conform to uniform reporting criteria. The greatest challenge lies in assessing minimal disease in extensive SLN evaluation. The success of the procedure is largely dependent on a multidisciplinary cooperation amongst surgeons, pathologists and nuclear medicine specialists. 14 Like any technique, frozen section has its own disadvantages including cost, labor, requirement of a skilled histopathology technician and a dedicated histopathologist for each surgical procedure. In a busy centre where multiple patients undergo operation in a single day, the burden on the histopathology staff and department can be immense. As a technique, frozen section is morphologically inferior to paraffin section histology (Fig. 3) and may miss subtle lymph node metastases (Fig. 4). Similarly, identification of the sentinel lymph node preoperatively using a blue dye or a radioactive isotope is associated with a number of problems ranging from logistical e like Table 3 Comparison of primary tumor size with proportion of sentinel nodal metastasis. Tumor size (cm) n ¼ 100 Proportion of micrometastases in sentinel nodes (%) No residual tumor 0 0 n ¼ 7 2 cm(t1) n ¼ % (n ¼ 1) 3.4% (n ¼ 1) >2 cm but 5 cm 16.4% (n ¼ 10) 34.4% (n ¼ 21) (T2) n ¼ 61 >5 cm (T3) n ¼ % (n ¼ 3) Proportion of macrometastases in sentinel nodes (%)

4 256 A. Moatasim et al. / International Journal of Surgery 11 (2013) 253e258 Table 4 Results of frozen section evaluation of SLN biopsy. Based on number of patients False positive 0 0 False negative 11% 8% Sensitivity (TP/TP þ FN) 90% 92.4% Specificity (TN/TN þ FP) 100% 100% Accuracy (TP þ TN/TP þ TN 96% 98% þ FP þ FN) Positive predictive value 100% 100% (TP/TP þ FP) Negative predictive value (TN/TN þ FN) 94.1% 97.4% Based on number of nodes handling radioactive material as well as tattooing e and rarely anaphylaxis caused by blue dye, not to mention failure to recognize the sentinel nodes by both techniques. A recent article has advocated the use of contrast enhanced ultrasound for correct preoperative identification of the sentinel lymph node, 15 biopsy of which if positive can lead to ALND without the need for intraoperative studies. According to Weiser et al. 6 the sensitivity of intraoperative frozen section is dependent on tumor size and false negative results are largely due to failure to detect micrometastases, a problem which can sometimes be addressed by performing rapid immunohistochemistry (IHC). A combination of touch imprint with either frozen section or rapid immunohistochemistry or the combination of all the three methods can help to improve the intraoperative diagnosis and allow detection of micrometastases. 16 Leikola and Weinberg 17,18 have recommended the use of intraoperative IHC to improve the intraoperative diagnosis of the SLN metastases. Although, IHC can be helpful in detecting micrometastases, its routine use in SLN assessment is controversial in terms of cost effectiveness and increased workload on the laboratory services. We did not perform immunohistochemistry intraoperatively in our study due to cost constraint. Its use was limited to one case on paraffin sections. In our study, the sensitivity, specificity, accuracy, and positive and negative predictive values of intraoperative sentinel lymph node assessment were 90%, 100% and 96%, 100% and 94.1% respectively based on number of patients. There was no false positive case in our study but the rate of false negative was 11% (Table 4). The 03/04 false negative cases on frozen section were those that harbored micrometastases missed because of thick nodal capsules not cut sufficiently finely. In 01/04 false negative case, a metastatic deposit was missed due to fat infiltration of lymph node that interfered with adequate cutting on frozen section. Understandably, serial slicing of lymph nodes, examining multiple levels and proven expertise of the histopathologist can improve the frozen section diagnosis. Another source of potential error not seen in our series is a lobular histology where cells are bland and have an infiltrative pattern. The reported sensitivity and false negative results of frozen section assessment of SLN biopsy by Veronesi et al. 19 were 64% and 24% respectively. Their high false negatives were due to micrometastases. Krag et al. 20 reported sensitivity, accuracy and negative predictive values of 88.6%, 96.8% and 95.7% respectively. In an eight year institutional study 21 on breast carcinoma patients undergoing intraoperative frozen section analysis of the SLN biopsy, the false-negative rate was 26.6%, the false-positive rate was Table 5 Comparison of diagnosis on frozen section vs permanent paraffin sections. Total no of sentinel Total sentinel nodes Total sentinel nodes lymph nodes removed positive on frozen section positive on paraffin sections Fig. 3. A large focus of metastatic carcinoma (arrow). Effacement of nodal architecture on the left by cords and sheets of tumor cells having pleomorphic, hyperchromatic nuclei (H&EX10M). Inset showing infiltrating ductal carcinoma Grade 2 on paraffin section of breast lump of same patient.. 0%, and the overall accuracy was 94%. Our study has shown significantly improved results due to meticulous sampling and examining multiple levels during frozen section. As in Veronesi s series, 19 the false negatives were all due to micrometastases and these patients were not offered further surgery, as per the protocol of the surgical unit. The size of a metastatic deposit is an important predictor of possible additional axillary metastases and can assist in personalizing surgical management of breast cancer. 22 In our study the size of nodal (sentinel) metastases ranged from 1 to 20 mm with a mean of 7 mm. The mean age of patients in our study was years, considerably younger than in Western countries. The mean tumor size in our study was 2.7 cm 1.3, ranging from 1.2 to 6.5 cm. The larger tumor size was due to multiple factors including late referral to a specialized breast centre due to lack of awareness, social pressures, limited financial resources, reluctance to be examined by male surgeons and shortage of female breast surgeons. The outcome can only be improved through a definite commitment by policy makers and health officials. Awareness campaigns are available but the efforts need to be exhaustive and a national drive for breast cancer screening is needed. These are only a few suggestions that can improve the outlook of breast carcinoma in our region. In future, automated molecular tests including quantitative reverse transcriptase-polymerase chain reaction (qrt-pcr) and one step nucleic acid amplification (OSNA) using mammoglobin (MGB) 1 and cytokeratin (CK) 19 can be utilized for detecting metastases in sentinel lymph nodes. Various papers have been published evaluating their application, many with promising results 23e27 with an overall sensitivity exceeding that of histopathological examination. These techniques also appear to detect lobular histology more effectively and can differentiate macro from micrometastases. These tests could be incorporated during frozen section evaluation of sentinel lymph nodes. Another future possible technique is elastic scattering spectroscopy (ESS) that detects the abnormal cellular architecture present in metastatic disease through changes in light absorption and scattering. ESS offers the possibility of intraoperative analysis of the sentinel node without the need for a specialist pathologist, and has other potential advantages such as minimal

5 A. Moatasim et al. / International Journal of Surgery 11 (2013) 253e Fig. 4. Micrometastasis (arrows) in a sentinel lymph node. (a) A suspicious focus seen on frozen section (H&EX4M), (b) On high power (H&EX20M) cords and groups of tumor cells seen in subcapsular area. tissue preparation and destruction, instant results and low running costs. Intraoperative analysis of the SLN biopsy continues to evolve, and is becoming more widespread. The question remains as to which technique will dominate future practice? Apart from conventional histology, intraoperative molecular-based techniques offer a greater ability to detect low-volume metastatic disease. They can provide quick results, are cost effective and do not invoke the expense of a dedicated pathologist. 28 As the debate continues, we should try to incorporate reliable, cost-effective techniques suited to our facilities and budget. 5. Conclusion We recommend routine frozen section evaluation of SLN biopsy in early breast carcinoma patients as it is a reliable and accurate technique allowing immediate assessment of axillary lymph node status at the time of surgery. However, it requires experience and is associated with pitfalls including failure to detect micrometastases. In skilled hands, the technique is almost comparable to routine histology and its advantages, including minimal morbidity and the avoidance of re operation with its associated costs; appear to outweigh its perceived drawbacks. Ethical approval As the article has no ethical issues, the study was approved by the head of department and ethical committee. Funding This is not a funded research. Author contribution Dr Ambreen Moatasim: Study design, data collection, data analysis, writing. Dr Shafaq Mujtaba: Data collection, writing. Dr Naveen Faridi: Data analysis, writing, review. Conflict of interest This article bears no conflicts of interest. 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