Characterizing an ideal experimental canine model for penile

1 Mestrado em Cirurgia, Universidade Federal do Ceará (UFC), Fortaleza, Ceará, Brasil. 2 Mestrando em Cirurgia Geral, Hospital Santa Casa de Misericordia de Sobral, Sobral, Ceará, Brasil. 3 Residência em Cancerologia Cirúrgica, Hospital Haroldo Juaçaba Instituto de Câncer do Ceará, Fortaleza, Ceará, Brasil. 4 Cirurgião Oncológico, Hospital Haroldo Juaçaba Instituto de Câncer do Ceará, Fortaleza, Ceará, Brasil. 5 Graduação em Medicina, Universidade Federal do Ceará (UFC), Fortaleza, Ceará, Brasil. 6 Doutorado em Cirurgia, Professor Titular pela Faculdade de Medicina, Universidade Federal do Ceará (UFC), Fortaleza, Ceará, Brasil.


INTRODUCTION
In Brazil, penile carcinoma (PC) is a rare neoplasm; representing only 2% of all malignant tumors diagnosed in Câncer (INCA) in 2013, 396 deaths were linked to PC whose main prevalence was in the north and northeast region. 1 Usually, PC disseminates via the lymphatic system by embolism rather than by lymphatic permeation.Furthermore, distant metastases are extremely rare and result from vascular dissemination. 2 Tumor staging is vital in programming treatment options, especially in patients with clinical disease but negative lymph node involvement.Prophylactic inguinal lymphadenectomy been shown to increase cure indices up to 80%, but it is marred with high levels of morbidity, psychological distress and functional disability.Unnecessary performed lymphadenectomy is associated with high levels of complications, among them hemorrhage, site infection, deep vein thrombosis and lymphedema. 3However, advances in sentinel lymph node (SLN) detection procedures have made it possible to identify patients with lymph node involvement.
Intra-operative gamma imaging (IGI) is widely used for SLN detection in patients with melanoma 4,5,6 and breast cancer. 7,8ue to its minimum invasive nature while at the same time highly sensitive, it is considered indispensable in SLN biopsy.
Anatomically, dogs are comparable to humans with regard to nodes in dogs are referred to as mammary lymph nodes as they drain the inguinal teats.They are located 3-4 cm cranially to the pubic bone on the dorso-lateral side of the mammary glands.Usually, two 0.5-2 cm long lymph nodes are observed, afferent lymphatic vessels of the skin and subcutaneous connective tissue of the abdomen, pelvis and hind legs are similar in males and females.
In an experimental study on female dogs, Pinheiro and contributors 9 found that patent blue dye (PB) and technetium 99m area, whether used alone or in combination.Using similar tracers, we intended to elucidate on inguinal lymphatic drainage in dogs.Therefore, our objective was to develop and evaluate an experimental canine model of penile sentinel lymph node detection using a combination of patent blue dye and technetium 99m phytate.

METHODOLOGY
After approval (06/08) by the animal research ethics committee of the Universidade Federal do Ceará (UFC), animals donated by the Municipal Zoonosis Control Center of Fortaleza (Ceará, Brazil) were sampled.The process randomly selected 19 adult male mongrel dogs (Canis familiaris) each weighing approximately 10-20kg.The animals were transferred and housed under controlled and comfortable environs (25°C, relative humidity approx.50%) of the Saul Goldenberg Laboratory (GEEON/UFC), with acess ad libitum to food and water.The circadian cycle was adhered to.Fasting was carried out 24 hours prior to the experiment.
During the experiment, a subcutaneous dose of 0.25mg/mL before an intramuscular injection of anesthesia (50mg/mLfor constant saline administration, using a 19G or 21G catheter inserted in the forepaw.Clinical parameters were constantly monitored through the experiment.Thereafter, using an insulin needle, 0.2 mL saline containing 5µCi technetium 99m was injected into the dermis of the penis glans along the mid rafe.After 10 99m Tc uptake by mapping the lymph-node mesh at the inguinal groove using a gamma probe, bilaterally.Next, patent blue dye was injected at the site of previous 99m Tc 99m Tc uptake at the inguinal objective was to identify the site with the highest uptake of 99m Tc. were made, guided by the intense presence of PB in the afferent lymphatic system until the SLN (or SLNs) was localized and way for the in vivo 99m Tc uptake and the level of staining.This was followed up by total lymph node dissection and resection for ex vivo 99m Tc uptake.A After quantifying the level of radiation and verifying that no more SNLs were existent at the inguinal lymph node groove, we performed hemostasis and sutured up the dissected region.After the experiment, the animal was euthanized at the surgical theatre with an intravenous bolus injection of 10% potassium chloride.

STATISTICAL ANALYSIS
The statistical analysis was performed with the software SPSS 20.0 (Statistical Package for the Social Sciences) license nº.10101131007.The data were analyzed with the McNemar test, the level of consistence between the two methods.

RESULTS
Table 1 shows lymph node 99m Tc uptake 5, 10 and 15 minutes after injection with technetium 99m and the respective standard deviations (SD).As shown in Table 2, the two sides (left/right) did 99m Tc uptake, PB staining, in vivo radiation count, ex vivo radiation count, or number of nodes stained with PB (p>0.05).The level of agreement between the two methods was 94.7% in vivo and 100% ex vivo.
The correlation between 99m Tc uptake and PB staining was 99m Tc as standard, a non-perfect correlation in vivo and a perfect correlation ex vivo was observed for the right side.The test was not applicable to the left side since all nodes were detected with the 99m Tc method (Tables 3 to 6).

DISCUSSION
The concept of sentinel lymph node (SLN) is well established, 10 although SLN biopsy in penile cancer remains controversial.][13] On the other hand, SLN biopsy is a widely validated procedure in breast cancer and skin melanoma.Basically, 14,15 Having this concept in mind, probably SLN in PC could help identify patients candidate for radical surgery.Consequently this would avoid unnecessary invasive procedures, reducing trans-and postoperative The administration of a radionuclide, usually by injection, and consequent lymphatic mapping using a gamma probe detecting SLN.Several authors 16,17 have evaluated the ability of 99m Tc-microaggregated albumin to identify SLNs by lympho-scintigraphy.A gamma probe was used to identify for exeresis of a respective SLN via a small incision.Currently, preoperative radionuclide lympho-scintigraphy is used to determine the location and quantify SLNs.Sometimes, PB 18,15   In our protocols, SLNs were detected in 94.76% of our cases, inguinal lymph node basins (p>0.05).As shown in Table 3 up to Table 6, PB and 99m Tc correlated well on the right side (kappa: 0.642) and perfectly on the left side (kappa: 1), indicating a high level of consistency.Our results are comparable to those reported by others.A review by Sapienza et al. on SLN detection in patients diagnosed with malignant melanoma using 99m Tc phytate and lympho-scintigraphy with 98.8% intra-operative detection. 19Similar results have been previously reported by us, though in dogs. 9Thus, the concept of SLN shows its consistency in studies, whether in canines or humans.
Anatomically, lymphatic drainage is much more predictable in PC than in melanoma, since the former has only two potential basin.The incidence of metastasis in patients with melanoma is approximately 1%, while that of PC is much less. 20,21In a study on patients with PC, Perdona et al. found similar rates of metastasis when comparing bilateral dissection to SLN biopsy (39% vs. 36%). 22However, the latter was associated with authors concluded in a recent review that when the metastases in the SLN are less than 2 mm, all other inguinal lymph nodes are almost certainly tumor-free and further dissection is unnecessary. 23e relevance of our study could be compared to that done by our group previously, which evaluated the ability of PB and 99m Tc phytate to identify SLNs in the subareolar region of the breast of female dogs. 9In that work, the authors found out that Up-to-date, this model is used by mastology residents at our institution to acquire skills in SLN detection in patients diagnosed with breast cancer.Furthermore, after evaluating and consequently validating the feasibility of our design on penile SLN -detection, this protocol could be applicable in the training of specialized surgeons.From an academic point of view, constant training could foster mastered skills, probably lead to a reduction in levels of false-negative results on inguinal SLN biopsy.
Studies involving SLN and PC are novel in literature elucidate continuous knowledge on this disease, which has a high prevalence in our region.Besides lower morbidity and improvement in quality of life of patients with PC, our protocols could serve as a benchmark for observation pattern. 24

CONCLUSION
Our results show that the experimental canine model of penile sentinel lymph node detection designed and evaluated in the present study is feasible.Patent blue dye and 99m technetium and were well correlated.The model may be used to test other methods of sentinel lymph node detection and adopted for clinical practice.

Table 1 .
Description of measures of central tendency and dispersion for Tc -99m uptake 5, 10 and 15 minutes after injection with Technetium -99m .

Table 2 .
Comparison of the right and left side with regard to method (technetium 99m and patent blue), and agreement between the methods.
**Not calculated, the variable was constant

Table 3 .
Level of agreement between the two methods of sentinel lymph node detection (technetium 99m vs patent blue) in vivo at the right inguinal groove.

Table 4 .
Agreement between the two methods of sentinel lymph node detection (technetium 99m and patent blue) in vivo for the left side.

Table 6 .
Agreement between the two methods of sentinel lymph node detection(technetium 99m and patent blue) ex vivo for the left side.