Ann Surg Oncol DOI 10.1245/s10434-014-4034-6

ORIGINAL ARTICLE – BREAST ONCOLOGY

Initial Results with Preoperative Tattooing of Biopsied Axillary Lymph Nodes and Correlation to Sentinel Lymph Nodes in Breast Cancer Patients Nicole Choy, MD1, Jafi Lipson, MD2, Catherine Porter, DO1, Michael Ozawa, MD3, Anne Kieryn, MD1, Sunita Pal, MD2, Jennifer Kao, MD2, Long Trinh, MD2, Amanda Wheeler, MD, FACS1, Debra Ikeda, MD, FACR2, Kristin Jensen, MD3, Kimberly Allison, MD3, and Irene Wapnir, MD, FACS1 1

Department of Surgery, Stanford University School of Medicine and the Stanford Cancer Institute, Stanford, CA; Department of Radiology, Stanford University School of Medicine and the Stanford Cancer Institute, Stanford, CA; 3 Department of Pathology, Stanford University School of Medicine and the Stanford Cancer Institute, Stanford, CA 2

ABSTRACT Background. Pretreatment evaluation of axillary lymph nodes (ALNs) and marking of biopsied nodes in patients with newly diagnosed breast cancer is becoming routine practice. We sought to test tattooing of biopsied ALNs with a sterile black carbon suspension (SpotTM). The intraoperative success of identifying tattooed ALNs and their concordance to sentinel nodes was determined. Methods. Women with suspicious ALNs and newly diagnosed breast cancer underwent palpation and/or ultrasound-guided fine needle aspiration or core needle biopsy, followed by injection of 0.1 to 0.5 ml of SpotTM ink into the cortex of ALNs and adjacent soft tissue. Group I underwent surgery first, and group II underwent neoadjuvant therapy followed by surgery. Identification of black pigment and concordance between sentinel and tattooed nodes was evaluated. Results. Twenty-eight patients were tattooed, 16 in group I and 12 in group II. Seventeen cases had evidence of atypia or metastases, 8 (50 %) in group I and 9 (75 %) in group II. Average number of days from tattooing to surgery was 22.9 (group I) and 130 (group II). Black tattoo ink was visualized intraoperatively in all cases, except one case with microscopic black pigment only. Fourteen group I and 10 group II patients had black pigment on histological examination of ALNs. Sentinel nodes corresponded to

Ó Society of Surgical Oncology 2014 First Received: 4 July 2014 I. Wapnir, MD, FACS e-mail: [email protected]

tattooed nodes in all except one group I patient with a tattooed non-sentinel node. Conclusion. Tattooed nodes are visible intraoperatively, even months later. This approach obviates the need for additional localization procedures during axillary staging.

Preoperative and pre-chemotherapy staging of the axilla with biopsy of suspicious nodes has become more common practice in patients with newly diagnosed breast cancer. Sonographic evaluation of the axilla has been incorporated into the imaging workup at many centers. Tagging of biopsied axillary lymph nodes (ALNs) with metal markers, similar to what is done for suspicious breast lesions, is being adopted in clinical practice.1,2 The need to mark a positive ALN becomes especially relevant in cases where neoadjuvant chemotherapy (NAC) is anticipated so that these nodes may be identified at the time of surgery. Measures that improve both the accuracy of nodal evaluation after NAC and the ability to assess treatment response are desirable in order to tailor therapies for breast cancer treatment.3 Ultrasound or mammography may not be well-suited to find metallic clips placed in the axilla, especially after neoadjuvant treatments. Identification may require computed tomography (CT) guidance with placement of a localization wire.4 More recently, 125I radioactive seeds and sonographically visible bio-resorbable polymers (HydromarkTM) have been promoted as alternative approaches.2,5,6 These labeling methods are disadvantaged by their resourceintensive and potentially more expensive methodologies involving radiologists either for placement or localization procedures.

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Because of the need to label relevant lymph nodes and avoid complex localization procedures at the time of surgery, we tested the feasibility of tattooing ALNs that are detectable months after injection and provide an inexpensive alternative to the existent method of placing metallic clips.2 The concept is based on the experience in the gastrointestinal tract wherein tattooing is widely used for marking lesions or tumors biopsied during endoscopy. India ink tattoos of colonic lesions remain identifiable over a long period of time.7 SpotTM is the first and only nonIndia ink FDA-approved product for marking or tattooing the gastrointestinal tract. It is a sterile, non-toxic, suspension containing water, glycerol, polysorbate 80, benzyl alcohol, simethicone, and high-purity carbon black.8 The primary aim of this study was to determine whether black ink tattoo injected into an ALN could be visualized intraoperatively in the same fashion blue sentinel nodes are identified. The second objective was to compare the preoperative pathological assessment with the histological findings of resected nodes, while the third objective was to determine the concordance between tattooed, biopsied ALNs and sentinel lymph node(s). We report our initial results on the feasibility of tattooing as an alternative method for marking ALNs in patients with breast cancer. METHODS Women aged 18 years or older with histologic evidence of breast cancer and clinically or sonographically suspicious ALNs were deemed eligible. Approval for the study was obtained from the Scientific Review Committee of the Stanford Cancer Institute and the Institutional Review Board. Written informed consent was obtained from all patients.

FIG. 1 Ex vivo photograph of tattooed axillary lymph node. a Macroscopic identification of tattoo pigment in lymph node cortex and adjacent soft tissue. b Small amount of tattoo ink seen macroscopically within the cortex of the lymph node or adjacent soft tissue may be missed during routine histologic sectioning

Tattoo Technique Intraoperative Evaluation The criteria used to select patients for lymph node biopsy was based on the presence of firm and/or matted nodes on palpation or the identification of abnormal nodes on ultrasound. The sonographic characteristics consist of finding generalized or focal thickening of nodal cortex, disparity in size of one or more lymph nodes compared with others, rounded appearance, and effacement of node fatty hilum.9 Palpation and/or ultrasound-guided fine needle aspiration (FNA) or core needle biopsy (CNB) was performed, followed immediately by injection of 0.1 to 0.5 ml of SpotTM ink into the cortex of the sampled lymph node and the adjacent soft tissue (Fig. 1). Patients with larger or palpable lymph nodes were injected with a greater amount of SpotTM ink. Patients were designated as—group I, surgery first cohort, and group II, NAC followed by surgery.

Two attending breast surgeons and trainees performed all the operative procedures, so the intraoperative observations were verifiable by more than one person. Sentinel lymph node procedures consisted of preoperative periareolar injection of technetium sulfur colloid (TSC) and/or intraoperative peritumoral injection of isosulfan blue dye. The axilla was inspected to determine whether black ink tattoo in node or soft tissue was visible (Fig. 2). Intraoperative touch-prep or frozen sections were performed on all sentinel lymph nodes in group II and the majority of patients in group I. Lymph nodes were routinely bisected, serially sectioned at 2 mm increments when larger than 0.4 cm thick. Sentinel and non-sentinel ALNs were examined using hematoxylin and eosin staining. All slides were also evaluated by one of three

Initial Results with Preoperative Tattooing

FIG. 2 Tattooed lymph nodes. a Blue lymphatic leading to sentinel lymph node stained with black tattoo pigment. b Microscopic identification of back tattoo pigment, 10X TABLE 1 Patient and tumor characteristics Group I (N = 16)

TABLE 2 Success of tattoo pigment identification Group II (N = 12)

Age (years)

55.6

54.0

Tumor size, cm (mean)

0.5–3.6 (1.9)

1.0–3.8 (2.7)

Clinical node status N0

5

4

N1

11

7

0

1

Positive node-pretreatment FNA/CNB False negative node FNA/CNB

N2

8 (50 %)

9 (75 %)

1 (6.2 %)

1 (8.3 %)

Mean number of SLNs per case

5.2

3.3

ER

13 (81.2 %)

8 (66.7 %)

HER2

2 (12.5 %)

2 (16.7 %)

Triple negative

3 (18.7 %)

3 (25.0 %)

Receptor status

FNA fine needle aspiration; CNB core needle biopsy; SLN sentinel lymph node biopsy

pathologists (KJ, MO, AK) in order to ascertain the presence of tattoo pigment either within the surrounding soft tissue or within the cortical surface of the node (Fig. 2).

Group 1 (N = 16)

Group 2 (N = 12)

Tattoo to surgery (range)

22.9 days (1–64)

130 days (74–211)

Intraoperative identification of tattoo pigment

15 (93.7 %)

12 (100 %)

Intraoperative mean # of tattooed nodes

1.5 nodes

1.6 nodes

Tattoo pigment

13

7

Intra-nodal alone

2

2

Adjacent soft tissue

3

1

Both

8

4

Tattooed node same as SLNB

14/15 (93.3 %)

12/12 (100 %)

Histology

SLNB sentinel lymph node biopsy

The injection of tattoo ink was performed by a breast surgeon in 17 patients, and by a radiologist in 11 patients. Cytological or histopathological evidence of nodal metastases was found preoperatively in eight (50 %) cases in group I, and pre-chemotherapy in nine (75 %) cases in group II. Overall, breast-conserving surgery was performed in 18 patients, and mastectomy in 10 patients. Intraoperative Visualization

RESULTS Twenty-eight patients underwent preoperative or pretreatment FNA or CNB and tattooing of ALNs. Mean age of the group I patients was 55.6 years, and 54.0 years for group II patients. Clinical tumor size ranged from 0.5 to 3.5 cm (mean 1.8 cm) in group I, and 0.5 to 3.8 cm (mean 2.7 cm) for group II prior to NAC (Table 1). All cases were clinically N0 or N1, except for one N2 case in group II.

The average number of days between tattooing and surgery was 22.9 days (range 1–62 days) for group I, and 130 days (range 74–211 days) for group II. No inadvertent tattooing of skin from the procedure was observed in any patient. Intraoperatively, black ink was identified in 15 of the 16 patients of group I and in all 12 of the patients of group II (Table 2). In the patient where no intraoperative black ink was identified, the histologic examination of the

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sentinel lymph node demonstrated evidence of black pigment below the cortex of the lymph node. Deeper injection into the node and the use of only 0.1 ml may explain the failure to intraoperatively visualize the tattoo in this case. The tattoo appeared localized to the sites of injection including perinodal soft tissue. Rarely, staining of an occasional lymphatic channel proximal to a node was observed. One patient in group II had a total of three tattooed nodes, one of which was high in level I of the axilla and the other two closer to the tail of the breast, demonstrating possible migration of the tattoo ink from one node to another node or staining of multiple nodes via a single injection. Sentinel Lymph Node Evaluation Lymphatic mapping and identification of sentinel nodes was carried out in all 28 patients, identifying, on average, 2.7 sentinel lymph nodes per patient. One patient in group I had a black-tattooed lymph node that did not corresponded to a sentinel lymph node and will be further described below. In group II, the tattooed lymph node corresponded to a sentinel lymph node in all cases. Axillary Ultrasound-Guided Fine Needle Aspiration or Core Needle Biopsy versus Surgery The preoperative axillary evaluation identified 8 of 16 patients in group I with nodal involvement by FNA or CNB. These were concordant with the final histopathological assessment. In one case, a cytologically negative node sampled by US-guided FNA corresponded with a histologically negative tattooed lymph node, but not with the histologically positive sentinel node. Axillary lymph node dissection (ALND) was performed in nine group I patients, eight (89 %) of whom were identified preoperatively as having nodal metastasis. Three patients were found to have additional disease in non-sentinel lymph nodes. Twelve patients in group II underwent definitive surgery after NAC, having successful lymphatic mapping and sentinel lymph node biopsy (SLNB) in all (Fig. 3). A total of five patients underwent completion ALND, four of whom had positive SLNB with findings of additional metastatic disease in non-sentinel lymph nodes. Six of nine patients with pretreatment involving positive ALNs were pathologically down-staged based on a negative SLNB. Of these, one patient proceeded to completion ALND, a decision influenced by clinical presentation criteria. One of the four patients who underwent ALND had a false negative pretreatment ultrasound-guided FNA. However, the black-tattooed node visualized intraoperatively was the involved sentinel node.

FIG. 3 Schematic of group II, neoadjuvant chemotherapy, FNA fine needle aspiration, CNB core needle biopsy, SLNB sentinel lymph node biopsy, ALND axillary lymph node dissection

Histologic Evaluation Histologic evaluation demonstrated the presence of microscopic black pigment in the cortical surface of the lymph node and/or in surrounding soft tissue in 14 patients of group I and 10 patients of group II (Table 2). Black pigment could not be identified on standard histologic sections in three cases, one in group I and two in group II, for whom tattoo ink was readily visualized intraoperatively. Conversely, black ink was microscopically detected in four of nine cases of group 1 who underwent axillary dissection. DISCUSSION Our initial results confirm that lymph nodes biopsied and tattooed with black ink during pretreatment diagnostic FNA or CNB can be identified intraoperatively. This represents an alternative approach for marking lymph nodes. Tattooed ALNs were identified in all patients treated with chemotherapy upfront. Intraoperative pigment was detected up to 211 days after injection, with an average time interval of 130 days. Other techniques have been described to mark positive ALNs, such as metallic markers and radioactive seeds.2,5,6 One advantage of the tattoo technique is that it does not require additional localizing procedures, thus decreasing cost and improving convenience. Although the distinction between the blue dye and black ink can be subtle, the tattoo ink displays a blackgreyish hue, while the isosulfan blue is an intense peacock blue (Fig. 2).

Initial Results with Preoperative Tattooing

The status of ALNs in breast cancer carries important prognostic information. Axillary ultrasound combined with FNA or CNB of suspicious nodes is helpful in the detection of axillary metastasis.10–12 The sensitivity and specificity of preoperative or pretreatment axillary ultrasound for metastatic lymph nodes ranges from 50 to 73 % and 70 to 96 %, respectively.13 The initial role was envisioned as a means of avoiding lymphatic mapping/sentinel node biopsy if a lymph node was positive.10,14–16 However, sampling of nodes has inherent limitations, as illustrated in a study of 110 patients with suspicious lymph nodes. Of the 36 samples with no evidence of metastases, 9 (25 %) were found to be node-positive on SLNB.17 Moreover, 146 patients with normal-appearing nodes on ultrasound were not sampled, and 21 (14 %) of these were found to have positive sentinel nodes.17 Surgical axillary staging remains the gold standard for accurate assessment of ALNs. Data from the ACOSOG Z0011 study has changed the approach toward axillary management, allowing the omission of an axillary dissection in select patients with clinically negative axillas and low burden of disease of one to two positive sentinel nodes.18 The role of sentinel lymph node dissection as an accurate assessment of axilla for nodal staging may be expanded based on emerging data. Sentinel node mapping and biopsy is gaining acceptance in the clinical scenario of neoadjuvant therapy. In a metaanalysis of 27 studies evaluating sentinel node procedures following NAC, the successful identification was 90.9 % (95 % confidence interval [CI] 88.0–93.1) and false negative rate (FNR) was 10.5 % (95 % CI 8.1–13.6).4 Pretreatment evaluation and biopsy of suspicious nodes to determine malignant involvement has been performed as part of various neoadjuvant studies.19–21 For clinically node-positive disease, lymphatic mapping and sentinel node biopsy after NAC has been evaluated in two large prospective trials—ACOSOG 1071 and the SENTINA trial.1,22 One of the interesting findings of the ACOSOG Z1071 trial was that the preoperative tagging of nodes was associated with higher accuracy and decrease in the FNR to 7.4 % (from 12.6 %), although not statistically significant.23 In our study, nodes identified as suspicious that were sampled and tattooed before treatment was initiated, correlated 96.4 % with those identified as sentinel nodes at the time of definitive surgery. Improving the accurate assessment of the ALNs after NAC in patients presenting with node-positive disease is critical for the selection of those who may forego completion ALND. Current National Comprehensive Cancer Network guidelines recommend preoperative axillary staging with ultrasound and FNA or CNB, along with placement of an image-detectable marker to be removed at the time of definitive surgery.24 Biopsy-proven positive

lymph nodes must be reliably excised and examined following NAC. The tattoo technique provides an easy method to verify the accuracy of preoperative pathological evaluation of nodes, requiring no additional imaging to either ascertain the location of the marker after placement or at the time of definitive surgery. Furthermore, the tattoo technique does not interfere with a standard SLNB and allows for correlation to pretreatment-biopsied lymph nodes. The amount of ink injected into the cortex of the node and the surrounding soft tissue ranged between 0.1 and 0.5 ml. As this report represents our initial experience, the optimal range of volume is being fine-tuned. In some cases, the ink seemed to diffuse more than expected into the soft tissue, whereas for larger nodes, increasing the volume of ink to 0.5 ml seems reasonable. Limitations to this technique include operator and site of injection variability. In three cases, black ink was identified intraoperatively, but not histologically. One explanation is that during processing, adipose tissue containing tattoo ink is dissected away from the surrounding lymph node, and is not captured on final histologic examination. Another possibility is that when the focus of tattoo pigment is small, it may not be captured within the level sampled histologically (Fig. 1); so that, the intraoperative identification of tattoo may be sufficient without the histological confirmation. CONCLUSIONS These data represent our initial experience with tattooing biopsied ALNs in women undergoing surgery first or NAC first. Tattooing is a feasible and low-cost method for marking biopsied nodes. The black pigment can be discerned by surgeons from blue dye and remains in place for months, as in the case of patients receiving NAC. In addition, this study demonstrates that sampled, tattooed lymph nodes correlate well with sentinel nodes in both groups, which add to the accuracy of surgical axillary staging. REFERENCES 1. Boughey JC, Suman VJ, Mittendorf EA, et. al. Sentinel lymph node surgery after neoadjuvant chemotherapy in patients with node-positive breast cancer. The ACOSOG Z1071 (Alliance) Clinical Trial. JAMA. 2013;310:1455–61. 2. Caudle AS, Cupp JA, Kuerer HM. Management of axillary disease. Surg Oncol Clin N Am. 2014;23:473–86. 3. Morrow M, Dang CT. Sentinel node biopsy after neoadjuvant chemotherapy, a new standard for patients with axillary metastases? JAMA. 2013;310(14):1449–50. 4. Bryant JA, Siddiqi NJ, Loveday EJ, Irvine GH. Presurgical, ultrasound-guided anchor-wire marking of impalpable cervical lymph nodes. J Laryngol Otol. 2005;119(8):627–8.

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