Intraoperative lymphatic mapping and sentinel lymphadenectomy has become an increasingly popular technique for staging the regional lymph nodes in early-stage melanoma. This operative technique allows for detailed pathologic analysis of the first (or sentinel) lymph node in direct connection with the primary tumor, and provides a unique opportunity for assessing potential immunologic interactions between the primary tumor and regional lymph node basin. We performed lymphatic mapping and sentinel lymphadenectomy on 25 patients with early-stage melanoma and resected an additional nonsentinel node in each case. Sentinel and nonsentinel nodes were evaluated by routine pathologic analysis. A portion of each node was processed for expression of the dendritic markers of activation CD80, CD86, and CD40, and their corresponding T-cell receptors CTLA-4 and CD28. Of 25 patients undergoing lymphatic mapping and sentinel lymphadenectomy, 20 (80%) had matched sentinel and nonsentinel nodes. A total of 26 matched lymph node sets were obtained: three pairs from one patient and two from an additional two patients. Reverse transcription polymerase chain reaction analyses of corresponding sections of the sentinel and nonsentinel nodes demonstrated a marked reduction in semiquantitative expression of CD80 (77%), CD86 (77%), and CD40 (85%), as well as CTLA-4 (88%) and CD28 (85%) in sentinel as compared to nonsentinel nodes. The diminished expression of the dendritic cell markers appeared to be unrelated to the B-cell (CD20) and T-cell (CD2) expression. Lymphatic mapping and sentinel lymphadenectomy allows for detailed pathologic and molecular characterization of sentinel nodes. Our results suggest a quantitative reduction in dendritic cell markers in sentinel as compared to nonsentinel nodes, which may be important in the immunologic interaction between the primary site and regional lymph node basin and may also serve as useful criteria for identifying sentinel nodes. [ONCOLOGY 16(Suppl 1):27-31, 2002]
The management of the regional lymph nodes in early-stage melanoma has been revolutionized by the development of lymphatic mapping and sentinel lymphadenectomy. This minimally invasive operative technique has replaced elective lymph node dissection and provides for precise identification and evaluation of the lymph nodes (sentinel node) in direct connection with the primary melanoma. Since the initial feasibility study by Morton and colleagues in 1992, multiple investigators have validated the accuracy of the technique and the ease with which this technology can be transferred to a variety of cancers, including colon and breast cancer.[1-3]
While lymphatic mapping and sentinel lymphadenectomy provides for detailed pathologic analysis of the sentinel nodes, it also allows investigators a unique opportunity to evaluate direct interactions between the microenvironment of the primary tumor and its relationship to the regional lymph nodes. Several clinical studies have suggested that physical alterations in the microscopic appearance of the regional lymph nodes resected concurrently with the primary tumor may be important for determining patient prognosis.[4,5]
Cochran and associates first described that both a physical and a functional alteration in the regional lymph nodes exist in early-stage melanoma. Prior to the development of lymphatic mapping and sentinel lymphadenectomy, his group demonstrated a differential response of proximal vs distal lymph node-derived lymphocytes from surgical specimens after in vitro stimulation with interleukin-2 and phytohemagglutinin in mixed lymphocyte reaction. These results suggested a diminished T-cell response from lymphocytes found in closer proximity to the primary melanoma.[6-8] The mechanism of this attenuated T-cell response at that time was unknown.
Recent studies have pointed toward the importance of dendritic cells as the initiating event in the immune response to malignancy. In melanoma, dendritic cell maturation, likely from passage of these cells from the skin to the regional lymph nodes, causes changes in the function of these cells from antigen processing to presentation. Associated with these changes are upregulation of the costimulatory molecule CD40 (a relatively early change) and the B7 molecules CD80 (B7.1) and CD86 (B7.2) necessary for T-cell activation.[10,11] Activation of dendritic cells occurs with promotion of T-cell maturation and expression of their corresponding receptors to the B7 molecules (CD28 and CTLA-4). The maturation of both dendritic and T cells is thought to be instrumental in the T-cell-driven response to tumor presentation.
While the mechanisms that control tumor-derived dendritic cell activation is yet unknown, the development of lymphatic mapping and sentinel lymphadenectomy provides a unique opportunity to analyze more specific relationships of dendritic cells directly connected to the primary (sentinel nodes) and those more distal to this site (nonsentinel nodes). Our hypothesis is that sentinel nodes would have a diminished expression of functional dendritic cell markers of activation as compared to nonsentinel nodes, based on the earlier studies demonstrating a suppressed T-cell response.
A total of 24 patients with American Joint Committee on Cancer (AJCC) stage I and II melanoma were considered for lymphatic mapping and sentinel lymphadenectomy after review of their pathology specimens and a thorough clinical exam demonstrating no sign of regional lymph node or distant metastases. No patient had a history of myeloproliferative disease or primary or secondary immunodeficiency.
All patients underwent lymphatic mapping and sentinel lymphadenectomy as previously described.[1,3] In brief, patients underwent preoperative cutaneous lymphoscintigraphy on the day of surgery. Patients were taken to the operating room after informed consent. In brief, lymphatic mapping and sentinel lymphadenectomy was performed with the combined use of blue dye and a radiopharmaceutical for probe-directed sentinel node biopsy. After excision of the sentinel node, the surgical wound was explored for adjacent, secondary, nonblue, nonradioactive, nonsentinel nodes. These nonsentinel nodes were usually identified within several centimeters of the sentinel nodes. In three cases, nonsentinel lymph nodes could not be identified. In two cases, the sentinel nodes were less than 1 cm in size and, as written in our protocol, we elected not to use tissue that may have been important for routine pathology review.
A total of 26 paired sentinel and nonsentinel nodes were analyzed, each pair from a separate lymph node basin. One patient had three pairs and two patients had two pairs. Fresh lymph nodes were immediately processed with complete freezing and tangential sections cut approximately 4 mm thick from both sides of the nodes parallel to the longest axis of the specimens. A single specimen was processed for reverse transcription polymerase chain reaction (RT-PCR) analyses. The remaining specimens were stored for other studies.
In brief, RNA was isolated from lymph node extracts using TRI Reagent (Molecular Research Center, Inc, Cincinnati). Total RNA was converted to cDNA with M-MLV reverse transcriptase and random hexamer (Promeda, Madison, Wisconsin). To assess the amount of mRNA of markers for dendritic cell activation, PCR was performed for CD80, CD86, CD40, CTLA-4, and CD28, and for the constituitively expressed housekeeping gene coding for GAPDH.
GAPDH primers (sense: 5¢-TGA AGG TCG GAG TCA ACG GAT TTG G-3¢; antisense: 5¢-GTT CAC ACC CAT GAC GAA CAT GG -3¢) were used as controls for semiquantification. CD2 and CD20 also served as quantitative B-cell and T-cell controls: CD2 (sense: 5¢-GGT CAT CGT TCC CAG GCA CCT AGT-3¢; antisense: 5¢-TGG TGT GAT GGA GCT CTC TGA GGA-3¢) and CD20 (sense: 5¢-CGT GCT CCA GAC CCA AAT CTA ACA-3¢; antisense: 5¢-GCG TGA CAA CAC AAG CTG CAA-3¢). Primers included CD80 (sense: 5¢-GTG GCA ACG CTG TCC TGT GGT-3¢; antisense: 5¢-CCA GGA GAG GTG AGG CTC-3¢), CD86 (sense: 5¢-CCA AAG CCT GAG TGA GCT AGT-3¢; antisense: 5¢-CTT AGG TTC TGG GTA ACC GTG-3¢), CD40 (sense: 5¢-TGG GGC TGC TTG CTG ACC GC-3¢; antisense: 5¢-CCA AAG CCG GGC GAG CAT GA-3¢), CTLA-4 (sense: 5¢-AGT ATG CAT CTC CAG GCA AAG C-3¢; antisense: 5¢-CCA GAG GAG GAA GTC AGA ATC TG-3¢), and CD28 (sense: 5¢-GTT TGA GTG CCT TGA TCA TGT GC-3¢; antisense: 5¢-GGC GAC TGC TTC ACC AAA ATC-3¢).
Amplification of 40 cycles was utilized in all samples, with PCR performed as previously described.[13,14] These conditions allowed sufficient linearity of PCR amplification. All PCR products were then separated by 2% agarose gel electrophoresis and detected by ethidium bromide fluorescence. Semiquantitation of band intensity was performed by comparison of densitometric readings of each band, correcting for GAPDH band intensity and background. Results are presented as ratios of sentinel node to nonsentinel node band intensity for each marker. Samples were run in triplicate to validate the accuracy of the results. A P value of less than .05 was considered significant.
A total of 25 consecutive patients were entered into the study. In three basins, nonsentinel nodes were not identified, and in two the lymph nodes were too small for analysis. Three patients had dual lymphatic drainage at lymphatic mapping and sentinel lymphadenectomy and another had drainage to three basins, for a total of 26 matched sentinel and nonsentinel node pairs from 20 patients. The primary melanomas had a mean depth of 1.46 mm (range, 0.30 to 3.30 mm). Four of the 20 patients (20%) were found to have metastatic disease in the sentinel node.
Laser densitometry was used to assess the relative gene expression of the dendritic- and T-cell markers as compared to the housekeeping gene GAPDH. A majority of the dendritic- and T-cell markers were expressed in lower levels in sentinel nodes as compared to nonsentinel nodes: CD80 (20/26, 77%), CD86 (20/26, 77%), CD40 (22/26, 85%), CTLA-4 (23/26, 88%), and CD28 (22/26, 85%) (Figure 1). Depressed expression of one marker always occurred along with depression of at least one other marker. Four patients had metastases to the sentinel nodes. In all four cases, gene expression was diminished in sentinel nodes as compared to nonsentinel nodes for each of the five markers. CD2 and CD20 marker expression was evaluated in all cases. The relative expression of these T-cell and B-cell markers was no different for sentinel and nonsentinel nodes (Figure 2).
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