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10.1245/ASO.2004.11.912
Annals of Surgical Oncology 11:174S-178 (2004)
© 2004 Society of Surgical Oncology
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SUPPLEMENT

Failure to Remove True Sentinel Nodes Can Cause Failure of the Sentinel Node Biopsy Technique: Evidence from Antimony Concentrations in False-Negative Sentinel Nodes from Melanoma Patients

Richard A. Scolyer, MBBS, John F. Thompson, MD, Ling-Xi L. Li, PhD, Alison Beavis, BSc, Michael Dawson, PhD, Phillip Doble, PhD, Vivian S. K. Ka, MD, J. Gregory McKinnon, MD, Robyne Soper, BSc, Roger F. Uren, MD, Helen M. Shaw, PhD, Jonathan R. Stretch, DPhil and Stanley W. McCarthy, MBBS

From the Sydney Melanoma Unit and Melanoma and Skin Cancer Research Institute (RAS, JFT, L-XLL, VSKK, JGMcK, RFU, HMS, JRS, SWMcC) and Department of Anatomical Pathology (RAS, RS, SWMcC), Royal Prince Alfred Hospital, Camperdown; Departments of Surgery (JFT, HMS, JRS) and Medicine (RFU), University of Sydney; and Department of Chemistry, Materials and Forensic Science (AB, MD, PD), Broadway, New South Wales, Australia.

Correspondence: Address correspondence and reprint requests to: Richard Scolyer, MD, Department of Anatomical Pathology, Royal Prince Alfred Hospital, Missenden Road, Camperdown, NSW 2050, Australia; Fax: 61-2-9515-8405; E-mail: richard.scolyer{at}email.cs.nsw.gov.au

ABSTRACT

We have recently found that antimony (originating from the technetium 99m antimony trisulfide colloid, used for preoperative lymphoscintigraphy) can be measured in tissue sections from archival paraffin blocks of sentinel nodes (SNs) by means of inductively coupled plasma mass spectrometry (ICP-MS) to confirm that removed nodes are true SNs. We performed a retrospective analysis of antimony concentrations in all our false-negative (FN) SNs to determine whether errors in lymphadenectomy (i.e., failure to remove true SNs) may be a cause of FN SN biopsies (SNBs). Among 27 patients with an FN SNB, metastases were found on histopathologic review of the original slides or additional sections in 7 of 23 patients for which they were available; however, antimony concentrations were low in 5 of 20 presumptive SNs. Our results suggest that an FN SNB can occur because of failure to remove the true SN as well as histopathologic misdiagnosis.

Key Words: False-negative • Melanoma • Regional recurrence • Sentinel node

The technique of sentinel node biopsy (SNB) has proved to be a highly accurate method of staging cutaneous melanoma.1–9 Furthermore, the presence or absence of metastases in the sentinel node (SN) is the most important prognostic factor for melanoma patients.10 Long-term follow-up of patients with negative SNs shows a small but definite incidence of recurrence in the mapped and sampled nodal basin.4,11–14 These patients may be considered to have a false-negative (FN) result of the SNB.

The assessment of SNs requires a team approach involving surgeons, nuclear medicine physicians, and pathologists. For an SN to provide accurate prognostic information, it is essential that "true" SNs are removed and examined thoroughly. Technical failures may occur as a result of errors in lymphatic mapping and sentinel lymphadenectomy or because of erroneous histologic evaluation. Previous reports have suggested that the majority of patients whose disease recurs in the relevant nodal basin will, in fact, have identifiable metastases in the biopsied SN if a more comprehensive evaluation of the node is undertaken.11,12 However, a review of the results of SNB procedures at the Sydney Melanoma Unit (SMU) has suggested that an incorrect histologic diagnosis has been responsible for only a minority of regional failures in SMU patients.15 One possible reason for this is that all specimens have had multiple sections and immunohistochemical stains used for diagnosis since the introduction of the technique at our institution.

The procedure of SNB involves preoperative injection of a technetium 99m (Tc-99m)-labeled colloid (Tc-99m antimony trisulfide colloid [99mTc-Sb2S3] at the SMU) for lymphoscintigraphy (LSG) and intraoperative injection of blue dye at the primary melanoma site for visual identification of SNs. The technique is demanding, and as it becomes more widely used, the training, skill, and competence of those performing it may be suboptimal.6,16–20 A technique to confirm true SN identity may reduce the FN rate of the procedure. We have recently shown that determination of antimony concentrations in tissue sections from archival paraffin blocks of SNs using inductively coupled plasma mass spectrometry (ICP-MS) is a highly sensitive and specific method for identifying true SNs.21 In the present study, we measured the antimony concentration in FN SNs in an attempt to determine whether failure to remove true SNs may be a cause of FN SNB results.

MATERIALS AND METHODS

Between March 1992 and June 2001, 1330 patients with a single primary cutaneous melanoma underwent SNB and were followed up at the SMU. Patients were offered an SNB in the context of the Multicenter Selective Lymphadenectomy Trial after April 1994 if their tumors were greater than or equal to 1 mm thick and/or of Clark level IV and showed no clinical signs of metastasis. Of these procedures, 157 were performed in conjunction with an elective lymph node dissection and were excluded from the analysis. An SN could not be identified in 21 patients and was found to be positive in 176. These patients were also excluded. Follow-up was incomplete for 19 patients, leaving 957 for analysis. An SNB result was defined as FN if the patient developed a first recurrence in the mapped nodal basin (25 cases) or if specimens were reexamined for any reason and found to be pathologically positive (two cases: one was reviewed because of a local recurrence and the other because of concern about extensive lymphovascular invasion in the primary tumor). Recurrences in the nodal basin following local or in-transit recurrence were not considered to be FN.

Lymphoscintigraphy
After histopathologic confirmation of the diagnosis of cutaneous melanoma in the primary lesion, LSG was performed in all except three of the 957 patients. Details of the protocol used at the SMU have been published previously.17,22 In brief, four intradermal injections of technetium 99m antimony sulfide colloid (particle size, 5 to 40 nm), each 0.05 to 0.1 mL in volume, were given around the primary melanoma site. Early and delayed imaging was then performed and the location of each SN was marked with a skin tattoo spot, and its depth beneath the skin surface was recorded. All node mapping was done after a biopsy confirming the diagnosis of melanoma but before definitive wide excision.

Operative Technique
The SNs were biopsied within 24 hours of the isotope injection. Approximately 15 minutes prior to the operative procedure, a total of 1.0 to 2.0 mL of Patent Blue V dye (Guerbert, Aulney-Sous-Bois, France) was injected intradermally at four to six sites around the biopsy scar. A careful search for each SN was conducted, guided by the lymphoscintigram and aided by blue dye visualization. The identity of the SN was confirmed by the presence of blue staining in the node. A hand-held gamma probe was not used in the identification of the SN in the first few years of the SMU experience. When it was used (since approximately April 1996), the SN was identified by radioactivity counts that were at least three times the residual count in the nodal basin.17 All nodes identified as SNs in the dynamic LSG report were sought and removed if possible. All patients were treated by wide excision of the primary site following the SNB. Patient data and follow-up information were entered into a prospectively collected database.

Histologic Assessment of Sentinel Nodes
SNs from all patients were cut along their longitudinal axes in 3-mm slices and embedded entirely in paraffin blocks following tissue processing. Four sequential 5-micron-thick tissue sections were cut from each block and stained with H&E (on sections 1 and 4) and with immunohistochemical markers for S100 and HMB-45 (on sections 2 and 3).

Analysis after Recurrence
In the histopathologic analysis, all histologic slides were reinterpreted by a single pathologist (RAS); if they were again negative, further sections were cut from each tissue block. The additional sections consisted of four serial sections obtained at each of two levels (50 microns apart). The sections from each level were prepared with H&E and with immunohistochemical stains for S100 protein, HMB-45, and melan A. Both the original and additional slides were examined microscopically by scanning of the entire slides at a magnification of 100 times.

For measurement of antimony concentrations, 50-micron-thick sections were cut from the tissue blocks of each FN SN with a Jung Biocut 2035 rotary microtome (Leica Microsystems Pty Ltd, Australia). The tissue sections were digested with microwave-generated heat as previously described,21 and the concentrations of antimony were measured with ICP-MS by means of a Sciex Elan 5100 (Perkin Elmer) with solution nebulization.21 All tissue samples were measured with no knowledge of whether they represented SNs or non-SNs. On the basis of the results of a previous study of antimony concentrations in 24 pairs of SNs and non-SNs removed from one regional site during the same operation from individual patients, an antimony concentration threshold for optimally defining the examined nodes as SNs or non-SNs was set at 0.11 ppb (unpublished data). The antimony level in all the FN SNs was determined to validate whether it was above or below the concentration threshold. When more than one SN was removed, each was examined but only the highest antimony level was recorded.

RESULTS

Of the entire series of 957 patients, 27 (2.8%) were found to have had an FN SNB result. Of the 27 patients with an FN result, 25 recurred in the mapped and sampled nodal basin and 2 had their original specimens reviewed for other reasons and were then found to be positive. The FN rate for the entire series (FNs/all positives) was 27 of 27 plus 176, or 13.3%.

Pathologic Failure
The original histologic slides of the SN were available for review for 22 of 27 patients. Two showed evidence of metastatic melanoma on histopathologic review; one was reviewed because of a local recurrence and the other because of concern about extensive lymphovascular invasion in the primary tumor. Five others demonstrated evidence of metastasis on reanalysis of the original block, with further sectioning and immunohistochemical staining of additional sections for S-100, HMB-45, and melan A.

Antimony Concentrations
The archival paraffin blocks of tissue of the SNs were available for sectioning for antimony determination for 20 of 27 patients. The concentrations of antimony in the FN SNs are presented in Table 1. Five of the 20 FN SNs had antimony concentrations of less than the concentration threshold (0.11 ppb) and probably represented non-SNs. The antimony levels of the SNs of all seven cases in which melanoma cells were detected on pathologic review were each greater than the concentration threshold, reconfirming that each of these was a true SN.


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  		TABLE 1. Concentration of antimony in 20 FN SNBs
 
Outcome for FN Patients
After a median follow-up of 21.5 months, 9 of the 27 patients with an FN SNB had died of their disease and 2 were alive with disease at last follow-up.

DISCUSSION

A small proportion of patients with a negative SN by histopathologic examination subsequently develop nodal metastases in the mapped nodal basin (2.8% of patients in this study).4,11–13,23,24 FN SNs can occur because of errors in lymphatic mapping, sentinel lymphadenectomy, or pathologic examination. A technique to determine whether the SN removed is a true SN has the potential to identify surgical causes of FN SNBs, i.e., those in which the failure may be attributable to failure to remove the true SN. Because the blue dye does not survive the processing procedures necessary for histopathologic examination and all the 99mTc has decayed by the time that sample is ready for analysis, these features cannot be used retrospectively to confirm that the true SN has been removed. Recently, Haigh et al. at the John Wayne Cancer Institute reported that carbon pigment can be used to confirm the identity of SNs in cutaneous melanoma.25 They found carbon particles in all tumor-positive SNs (n = 18) and 90% of tumor-negative SNs (n = 93). A potential problem with this technique is that the presence of the large, dense carbon particles may obscure the identification of metastatic tumor cells within the SN and result in an FN pathologic diagnosis.

We have recently found that measuring antimony concentrations by ICP-MS is a highly sensitive and specific method of differentiating SNs from non-SNs (nodes removed from patients without melanoma).21 This technique has a great advantage over the carbon particle technique described by Haigh et al.25 in that it can be employed within existing pathologic practices used to examine SNs. In a further study using ICP-MS on paired SN and non-SNs removed from the same regional lymph node field of individual patients during the same operative procedure, we found that this technique was highly accurate in differentiating SNs from non-SNs, with sections of SNs containing significantly greater antimony concentrations than those from non-SNs (P = .004) (R.A. Scolyer, unpublished data, November 2002).

The antimony concentrations in the SNs of all seven patients in whom metastatic melanoma cells were identified on pathologic review were high, consistent with each being a true SN and indicating that the cause of the FN SNB was pathologic failure. The finding that five FN SNs had very low antimony levels suggests that the cause in these cases may have been removal of the wrong node (surgical failures). In the remaining 15 patients the cause of the FN SNB remains unclear. Because only a portion of the total nodal tissue of each SN was examined pathologically, we cannot exclude the possibility that occult melanoma cells may have been present in parts of the SNs that were not examined. Other possible causes of FN SNBs include failure to identify all the SNs by lymphoscintigraphy (failure due to error in lymphatic mapping) or failure to remove all SNs surgically when more than one SN was present.

It is interesting that four of the five cases with low antimony concentrations in the presumptive SNs were patients treated before April 1996. A hand-held gamma probe was not used to identify these SNs; they were identified intraoperatively only by the presence of lymphatic channels containing blue dye entering the nodes and by observing blue staining of the nodes themselves. The very much lower frequency of surgical failures after the introduction of the hand-held gamma probe at the SMU in 1996 suggests that the identification of SNs by a combination of visualization of blue dye and radioactivity counts guided by preoperative LSG is a more accurate method than the use of blue dye alone. Other factors such as increased experience and familiarity with the technique may also have contributed to the reduction in surgical failures.

The five procedures that resulted in the low-antimony-concentration FN SNBs were performed by three different surgeons (two surgeons each performing two of the FN SNB procedures and one surgeon the remaining one). All three surgeons had extensive experience in SNB, and the fact that the failures were spread among them suggests that a systematic error in technique was not the cause of the failures.

Antimony concentrations of appropriately prepared samples can be determined by ICP-MS within a few minutes. Thus, this technique has the potential to provide a rapid and reliable method of confirming the identity of true SNs intraoperatively and could thereby reduce the FN rate of the SNB technique. The results of our preliminary studies suggest that it may be possible to measure antimony concentrations by examination of material obtained from presumptive SNs, with use of a minimally invasive fine-needle aspiration biopsy technique, under ultrasound control.

CONCLUSIONS

An FN SNB for patients with melanoma is an unusual event but one that may have very serious implications. Quantitation of antimony concentrations within SNs by means of the highly sensitive technique of ICP-MS can confirm the identity of SNs and validate the SN technique. Our results suggest that an FN SNB can occur because of failure to remove the true SN as well as histopathologic misdiagnosis. Potentially, determination of antimony concentrations of fine-needle aspirate biopsies could be used to confirm the identity of SNs intraoperatively.

ACKNOWLEDGMENTS

The authors thank Marjorie Colman for invaluable assistance in managing and analyzing the SMU database. This study was supported in part by the Fulbright Scholarship fund, the Walter C. MacKenzie–Scotiabank Fellowship, the Royal College of Pathologists of Australasia, the University of Sydney Cancer Research Fund, and the Sydney Melanoma Foundation.

The acknowledgments are available online in the fulltext version at www.annalssurgicaloncology.org. They are not available in the PDF version.

FOOTNOTES

Antimony concentrations in all false-negative sentinal nodes (SNs) were retrospectively analyzed to determine whether errors in lymphadenectomy (i.e., failure to remove true SNs) might be a cause of false-negative SN biopsies. Results suggest that a false-negative SN biopsy can occur because of failure to remove the true SN as well as histopathologic misdiagnosis.

Received for publication October 27, 2003. Accepted for publication December 10, 2003.

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