Evaluation of tumor load in sentinel lymph node in patients with cutaneous melanoma

ABSTRACT Introduction: cutaneous melanoma (MC) is a malignant neoplasm derived from melanocytic cells with an aggressive behavior. It is usually associated with the multifactorial interaction of genetic susceptibility and environmental exposure, usually ultraviolet radiation. Despite advances in treatment, the disease remains relentless with poor prognosis. Sentinel lymph node (SLN) biopsy is a technique used to screen patients in need of lymph node dissection. Objectives: to correlate the tumor burden in the SLN with the mortality of patients undergoing SLN biopsy. Methodology: the medical records and histological slides of patients with MC who underwent SLN biopsy treated at HC-Unicamp from 2001 to 2021 were retrospectively analyzed. The positive SLN were measured according to the size of the tumor infiltration area, for analysis of the depth of invasion (DI), closest proximity to the capsule (CPC) and tumor burden (TB). For statistical analysis, associations between variables were analyzed using Fishers exact test, with post Bonferroni test and Wilcoxon test. Results: 105 records of patients who underwent SLN biopsy of MC were identified. Of these, nine (8.6%) had positive SLN and 81 (77.1%) had negative SLN. The performed lymphadenectomies resulted in 55.6% (n=5) affected, 22.2% (n=2) without disease and 22.2% (n=2) were not performed. Mean CPC, TB, and DI were 0.14mm, 32.10mm and 2.33mm, respectively. Patients with T2 and T3 tumors were more likely to show the SLN affected (p=0.022). No patient with positive SLN died during follow-up. Conclusion: patients who presented T3 staging are the ones who most presented positive SLN.


INTRODUCTION
C utaneous melanoma is a malignant neoplasm that is often aggressive and caused by melanocytic cells, responsible for producing melanin, the substance that determines skin color 1 .The lesion is multifactorial, being caused by the interaction between genetic susceptibility and environmental exposure, such as mainly sun exposure, artificial tanning, fair skin, increased number of melanocytic and/or dysplastic nevi, freckles, and family history of the disease 2,3 .Melanoma is estimated to constitute less than 5% of skin cancers, but it is responsible for about 95% of deaths from cancer in this organ, being the most severe skin cancer 5 .In Brazil, data from the National Cancer Institute for the year 2020 indicated melanoma incidence rates of 4.03 new cases per 100,000 in males and 3.94 new cases per 100,000 in females 4,5 .The main protective factor for the reduction of mortality is early diagnosis, the prognosis being directly proportional to the invasion in the depth of the skin 6 .
Sentinel lymph node (SLN) biopsy is a technique that aims to avoid aggressive lymphadenectomy surgeries that can lead to limiting complications for patients, such as limb lymphedema and neurovascular lesions.Conceptually, the technique consists of marking the SLN with lymphoscintigraphy prior to surgery, and intraoperatively locating it with a portable gamma radiation detector.Subsequently, the SLN is analyzed by the pathologist for the presence of metastasis.Once negative, the ganglionic chain can be considered free of metastasis and would avoid additional procedures 7 , being considered of high relevance for risk assessment and treatment strategy 7 .In this study, we aimed Pedro deak de almeida 1 ; luccas lavareze 2 ; carolina emerick da silva rangel 2 ; Fernanda viviane mariano 2 ; diego victor nunes rodrigues 3 ; tiago antonio Baldasso 1 ; renato ventura Fanni 1 ; andre luis maion casarim 1 ; andré del negro 1 ; alFio José tincani 1 .with sufficient tissue for histopathological evaluation and subsequent analysis.We excluded patients who had previously been treated or manipulated in the SLN drainage area.
In the analysis of medical records, we collected data regarding age, color, race or ethnicity, sex, primary location of the melanoma, Breslow index of the primary melanoma, T staging of the primary melanoma, and survival.We divided patients into positive and negative SLN groups for the analyses.
Two examiners analyzed the H&E slides with their respective diagnoses to confirm the presence or absence of metastasis in the sentinel lymph nodes.
Photomicrographs of positive SLNs were taken using five-and ten-fold augmentation lenses, according to the size of the tumor infiltration area, for analysis of depth of invasion (DI), closest proximity to the capsule (CPC), and tumor burden (TB).The photomicrographs were taken using a Leica ® microscope (Leica Microsystems, Switzerland) and processed using the Leica ® software, version 4.2.0 (Leica Microsystems, Switzerland).We defined DI as the largest area of melanoma infiltration in millimeters (mm) inside the lymph node (Figure 1A).The CPC corresponded to the shortest distance (mm) between the tumor and the inner portion of the lymph node capsule (Figure 1B).Finally, TB was the sum of all metastatic areas in square millimeters (mm 2 ) in the lymph nodes (Figure 1C).Cases with more than one metastasis focus were photographed and evaluated in several fields, with the tabulation of the value corresponding to the definition of its variable (DI or CPC).We analyzed the images in the ImageJ software (National Institutes of Health, USA), version 1.53v, using a millimeter ruler photographed in the 5x or 10x lenses for standardization.

RESULTS
We identified 105 records of patients who underwent a melanoma SLN biopsy in the study period.
This number was reduced because in the period between 2004 and 2006 this procedure was not performed due to operational problems.Sample demographics are described in Table 1.The sample had a mean age of 54.1 ± 14.0, and a similar distribution between sexes, 50.5% (n=53) women and 49.5% (n=52) men.Most patients were white (94.3%; n=99), with tumors on the trunk (38%, n=40) and upper limbs (30.4%, n=32).As for the distribution of the tumor by sex, four (7.5%) cases were found in women in the head and neck, in contrast to seven (13.5%) in men.In the trunk, we identified 20 Almeida Evaluation of tumor load in sentinel lymph node in patients with cutaneous melanoma cases (37.7%) in women and 20 (38.4%) in men.We found Melanoma in the upper limbs in nine (16.9%) female cases and in 13 (25%) males.Involvement in the lower limbs happened in 20 (37.7%) women and in 12 (23%) men.TB: tumor burden.

DISCUSSION
Cutaneous melanoma (CM) is an aggressive malignant neoplasm that can have an unfavorable clinical course when diagnosed in late stages 2,3 .Although early diagnosis is the best way to improve prognosis, treatment for this tumor is still challenging 6 .Despite advances in drug therapy with BRAF inhibitors 8 , surgery methodology that points to a positive SLN at risk.The Rotterdan criterion states that the larger the TB, the greater the chance of involvement of the ganglionic chain, and SLNs with TB greater than 1mm could benefit from a complete dissection 18,19 .However, it is not uncommon for other methodologies to be found in the literature.Despite the heterogeneity of methods and results, there is a consensus that the chance of involvement of non-sentinel lymph nodes is proportional to the size of the TB in the SLN 20,21 .
Evidences point out that patients submitted to SLN biopsy present a better survival than those who remained under lymph node surveillance 11  In this sample, a reduced number of patients had a positive SLN (9%).In the literature, the general positivity rate ranges from 12% to 26% [13][14][15]

Introduction:
cutaneous melanoma (MC) is a malignant neoplasm derived from melanocytic cells with an aggressive behavior.It is usually associated with the multifactorial interaction of genetic susceptibility and environmental exposure, usually ultraviolet radiation.Despite advances in treatment, the disease remains relentless with poor prognosis.Sentinel lymph node (SLN) biopsy is a technique used to screen patients in need of lymph node dissection.Objectives: to correlate the tumor burden in the SLN with the mortality of patients undergoing SLN biopsy.Methodology: the medical records and histological slides of patients with MC who underwent SLN biopsy treated at HC-Unicamp from 2001 to 2021 were retrospectively analyzed.The positive SLN were measured according to the size of the tumor infiltration area, for analysis of the depth of invasion (DI), closest proximity to the capsule (CPC) and tumor burden (TB).

Figure 1 .
Figure 1.DI, CPC, and TB measurement methodology.For statistical analysis, we assessed associations between variables using the Fisher's exact test, with Bonferroni post-test and Wilcoxon test.All analyzes were performed using the R software (R Foundation for Statistical Computing, Vienna, Austria) with a significance level of 5%.
For statistical analysis, associations between variables were analyzed using Fishers exact test, with post Bonferroni test and Wilcoxon test.Results: 105 records of patients who underwent SLN biopsy of MC were identified.Of these, nine (8.6%) had positive SLN and

Table 1 -
Demographic characteristics of the sample, 2022.
of patients were lost to follow-up in the last three years, in addition to 5.7% of patients' records not showing any follow-up.In the group of patients with positive SLN, there was a 44% loss to follow-up, with a mean survival until the last visit of 2.2 years.The average survival of the followed patients was 10.79 years.Tabela 2 -Sample characteristics according to the presence of positive SLN.n=79: 7 positive SLN; 72 negative SLN; † n=79; * Statistical difference between groups; DI: depth of invasion; CPC: closest proximity to the capsule;

Table 3 -
Histological measurements of positive SLN.
DI: depth of invasion; CPC: closest proximity to the capsule; TB: tumor burden.
Therapeutic decisions regarding the positivity of an SLN are still discussed in the literature.The absence of standardization and criteria to quantify the tumor invasion load in SLN makes it difficult to define a