Preoperative planar lymphoscintigraphy allows for sentinel lymph node detection in 51 dogs improving staging accuracy: Feasibility and pitfalls

Abstract Sentinel lymph node (SLN) mapping is the current gold standard for the oncological staging of solid malignancies in humans. This prospective observational study describes the feasibility and the limits of preoperative lymphoscintigraphy for SLN detection in dogs with spontaneous malignancies and the improvements in staging accuracy. Client‐owned dogs with confirmed malignant neoplasia and absence of distant metastasis were prospectively enrolled. Lymphoscintigraphy was performed after the peritumoral injection of Technetium‐99m labeled nanocolloids. Regional dynamic and static images were acquired, with and without masking of the injection site with a lead shield. The dogs were then subjected to surgery for tumor excision and SLN extirpation. Intraoperative SLN detection was performed by combining methylene blue dye and a dedicated gamma probe. Overall, 51 dogs with a total of 60 solid malignant tumors were enrolled. Lymphoscintigraphy identified at least one SLN in 57 of 60 cases (95%). The SLN did not always correspond to the regional lymph node (35/57, 61.4%). The use of a lead shield, masking the injection site, markedly improved the SLN visibility. The median time of SLN appearance was 11.4 ± 9.3 min. No side effects were observed. Preoperative lymphoscintigraphy allows for SLN detection in dogs and can improve staging accuracy by either identifying the SLN in a different lymphosome than clinically expected or discriminating the draining node in uncertain cases. The combined use of preoperative and intraoperative techniques is recommended to increase the SLN detection rate.


INTRODUCTION
The sentinel lymph node (SLN) is the first lymph node (LN) that receives direct lymphatic drainage from a primary tumor site. 1 In veterinary medicine, sampling the regional lymph nodes (RLN), only when palpably enlarged, is notoriously inaccurate, whereas sampling an inappropriate LN might lead to false-negative results and thereby understaging. 2,3,4 Tumor-induced lymphangiogenesis increases the variability of lymphatic drainage patterns, thus hampering the possibility of SLN identification based on anatomical location alone. 5 Therefore, SLN mapping procedure indicates the correct LN to be assessed for the presence of nodal metastasis, allowing a more accurate tumor staging, thus aiding treatment decision and prognostication. [6][7][8][9] Furthermore, a potential therapeutic role of early nodal intervention in oncological patients with no signs of lymphadenopathy has been hypothesized, although it remains to be investigated in both, human 10 and canine 11 oncology.

Different mapping techniques have been developed in human and
veterinary oncology based on the same principle i.e., to inject a tracer at the primary tumor site and follow the lymphatic pathways to the first draining LN. 12,13,14,15,16 Methylene blue (MB) was the first tracer used for lymphadenography in patients with penile carcinoma. 17 Radiocolloids, associated with blue dyes, were first used in human oncology for SLN mapping in patients with melanoma 18 and, among other malignancies, the breast 19 and prostate 20 cancer. Radiocolloids for SLN mapping includes preoperative planar lymphoscintigraphy as well as intraoperative nodal detection with a hand-held gamma probe. The most common radionuclide is the Technetium-99m (99m−Tc), which has a physical half-life of 6.01 h and emits gamma (photons) rays between 140.5 keV (98.6%) and 142.6 keV (1.4%). 21 Colloids used for radionuclide labeling includes the sulfur colloid (<200 nm), human serum albumin nanoparticles (<80 nm), and antimony sulfide colloid (10 nm

Patients
In this prospective observational study, a convenience sample without a predetermined cohort size was enrolled. Client-owned dogs admitted to a veterinary institution for oncological surgery between June 2016 and October 2019 and meeting the inclusion criteria were included.
A part of the patients (30 dogs) was included in a study concerning the impact of SLN biopsy on oncological staging in dogs with mast cell tumor. 6 The inclusion criteria were as follows: (a) cytological or his-  For systemic side effect, during the general anesthesia, the cardiovascular and pulmonary parameters were checked and reported on the anesthesiology record, any unpredictable changes had been described.

Intraoperative SLN detection
After planar lymphoscintigraphy, the animals were admitted to surgery  colleagues. 26 Sentinel lymph nodes detected with lymphoscintigraphy, gamma probe, and MB were recorded and compared to the RLNs reported to normally drain the tumor's anatomical territory. 26 Other recorded data included the effect of the IS masking on SLN visibility during lymphoscintigraphy, the presence or absence of histopathologic LN metastasis, radiotracer immediate and delayed side effects.

Patients
Fifty-one dogs were enrolled in the study. The ages of dogs ranged

Sentinel lymph node detection
Lymphoscintigraphy located the SLN in 57 of the 60 tumors (95%).

Side effects
Twelve dogs with MCT showed mild swelling at the site of radiopharmaceutical injection. Duration of swelling was not evaluated due to the immediate admission of the patients to surgical extirpation of tumor. Both dogs with oral melanoma and the dog with sub-lingual SCC showed minimal bleeding related to the injection procedures, which was resolved immediately with direct pressure hemostasis.

DISCUSSION
This prospective study demonstrates the feasibility, safety, and effec- In our investigation, one dog had a palpable thyroid carcinoma and received a peritumoral radiotracer injection that showed local activity diffusion without any lymphatic vessels or node detection. The second patient received an intratumoral ultrasound-guided radiotracer injection, as described in human oncology, 27  Early dynamic images in the preoperative lymphoscintigraphy protocol did not offer additional clinical information on SLN detection, compared to static images, which is in line with previous research in human literature. 34 After the first appearance of the SLN on the dynamic or static images, orthogonal views need to be combined to correctly identify the SLN basin. To overcome the lack of anatomical details provided by lymphoscintigraphy, a syringe with a residual radiopharmaceutical was used as a pointer, as previously described. 3,25 Under the guidance of real-time images displayed on the gamma cam- can also lead to a challenging visualization of the LN uptake ("shinethrough effect" 35 ). The use of a 2 mm lead shield to cover the IS subjectively improved the SLN visibility during the procedure and reduced the concealing of the highly radioactive site on nearby nodes.
However, in two dogs (n. 16 and 28) with an MCT of the right stifle, the intraoperative gamma probe identified an additional radioactive LN in the popliteal basin, which was not identified with lymphoscintigraphy, probably due to superimposition between the IS and the LN.
In this study, the intraoperative use of a gamma probe allowed a targeted identification of radioactive SLN included in the lymphatic basin detected by preoperative planar lymphoscintigraphy.
In the dog presenting with sub-lingual SCC (patient n. 33), MB identified an additional LN in the contralateral mandibular basin, which was not radioactive but was metastatic. In human oncology, the failure of SLN identification is associated with an increased risk of metastases to the axillary lymph system. 29 Recently, Rossi et al 36  The results confirm the hypothesis that the first LN draining the tumor site could be identified within unexpected lymphatic basins. As previously stated, 6,8,9 SLN mapping is a keystone concept in canine oncological staging, identifying the correct LN to sample in order to improve patient prognostication and adjuvant therapy selection.
An important limitation of scintigraphy is the need for authorization and equipment for radioactive isotope use, which limits the number of facilities with nuclear medicine services. In addition, specific precautionary procedures are required to guarantee personnel safety, despite radiation exposure being reported to be minimal. 8 As a consequence, other SLN mapping techniques have been developed in human medicine and further described for oncological staging purposes in dogs, such as the radiographic lymphography, 35 CT lymphography, 14,15,36,37,38 contrast-enhanced ultrasound, 16,39 and near-infrared imaging. 40,41 In this study, we decided to perform SLN extirpation and histopathological assessment without prior nodal sampling. This was motivated by the reported low sensitivity and specificity of fine needle cytology compared to histopathology 42 in assessing LN metastatic status, and, in dogs with MCT, by the importance of histological lymph node categorization 7 and the hypothesized therapeutic role of lymphadenectomy. 11,43 The long-term effects of removing normal LN is still unknown and worthy of further investigations.
In conclusion, preoperative planar lymphoscintigraphy is a feasible, safe, and effective method for SLN detection in dogs with spontaneous malignancies in different anatomical locations. This mapping technique drives the surgical extirpation of SLNs in a specific lymphocentrum, allowing for accurate oncologic staging, either for identifying the tumor-draining node within unexpected lymphatic basins or discriminating the draining node in uncertain cases. The use of combined preoperative and intraoperative techniques is recommended to improve the SLN detection rate.

ACKNOWLEDGMENT
This study was supported by "Piano strategico per il sostegno alla ricerca Linea 2 -Azione A 2017″.