False-positive cells in sentinel lymph nodes

Melanoma sentinel lymph nodes (SLN) are carefully evaluated to maximize sensitivity. Examination includes hematoxylin and eosin (H+E) stained sections at multiple levels through the node, with subsequent immunohistochemical (IHC) stains for melanocytic markers if H+E sections are negative for melanoma. However, not all IHC-positive cells in SLN are metastatic melanoma, as evidenced by the presence of MART-1 positive cells in SLN from breast cancer patients with no history of melanoma (so-called 'false-positive' cells). These 'false-positive cells' could be nodal nevus, non-melanocytic cells with cross-reacting antigenic determinants, phagocytic cells containing melanocyte antigens, or possibly melanocytes or melanocyte stem cells liberated at the time of biopsy of the cutaneous melanoma. Examination of SLN requires careful correlation of H+E and IHC findings.     

Can Melan-A replace S-100 and HMB-45 in the evaluation of sentinel lymph nodes from patients with malignant melanoma?

The sentinel lymph node (SLN) biopsy has become an increasingly important procedure used in the primary staging of malignant melanoma. However, micrometastases in a lymph node can be easily missed on routine H&E-stained sections. Therefore, S-100 and HMB-45 IHC stains are standardly performed on grossly negative SLNs for detection of metastatic melanoma. Each of these IHC markers, however, is not ideal. The authors investigated whether the newer IHC marker Melan-A would improve the detection of metastatic melanoma in SLN biopsies. Forty lymph nodes previously diagnosed with metastatic melanoma were retrospectively evaluated for S-100, HMB-45, and Melan-A expression. In addition, 42 SLN biopsies for metastatic melanoma detection were prospectively collected and evaluated for S-100, HMB-45, and Melan-A expression. All lymph nodes with metastatic melanoma from the retrospective study demonstrated S-100 reactivity. Five of the lymph nodes with metastatic melanoma from the retrospective study failed to express either HMB-45 or Melan-A, all of which displayed a desmoplastic morphology. One of the metastases positive for S-100 and HMB-45 failed to show reactivity with Melan-A (3%). The prospective study found 10 lymph nodes from 42 cases to be positive for metastatic melanoma, which were positive for S-100 (100%). Nine of the involved lymph nodes were positive for HMB-45(90%), and nine were positive for Melan-A (90%). Melan-A, although very specific, cannot replace the use of S-100 and HMB-45 for the detection of metastatic melanoma in SLNs. It can, however, substitute for HMB-45 with equally good results.     

Histopathological patterns of melanoma metastases in sentinel lymph nodes

AIMS: Sentinel lymph node biopsy (SLNB) is an important component in the staging and treatment of cutaneous melanoma (CM). The medical literature provides only limited information regarding melanoma sentinel lymph node (SLN) histology. This report details the specific histological patterns of melanoma metastases in sentinel lymph nodes (SLNs) and highlights some key factors in evaluating SLNs for melanoma. METHODS: From 281 SLNB cases between June 1998 and May 2002, 79 consecutive cases of SLN biopsies positive for metastases from CM were retrospectively reviewed. The important characteristics of the SLNs and the metastatic foci are described. RESULTS: The median size of positive SLNs was 17 mm (range, 5-38). SLNs had a median of two metastatic foci (range, 1-11), with the largest foci being a median of 1.1 mm in size (range, 0.05-24). S-100 and HMB-45 staining was positive in 100% and 92% of the detected metastatic foci, respectively. The metastatic melanoma cells were epithelioid, spindled, and mixed in 86%, 5%, and 9% of cases. Metastatic foci were most often (86%) found in the subcapsular region of the SLN. Benign naevic cells were found coexisting in 14% of positive SLNs. CONCLUSIONS: Staining for S100 is more sensitive than HMB-45 (100% v 92%), but HMB-45 staining helped to distinguish benign naevic cells from melanoma. The subcapsular region was crucial in SLN evaluation, because it contained the metastases in 86% of cases. Evaluation of the subcapsular space should not be compromised by cautery artefacts or incomplete excision of the SLN.     

Anatomo-pathologic study of sentinel lymph nodes in melanoma. Analysis of 200 cases

Pathologic evaluation of sentinel lymph node represents a new technique for managing high-risk primary melanoma. We examined the sentinel lymph node biopsies of 200 patients affected by primary melanomas of trunk, limbs, head and neck, who had been operated at "M. Bufalini" Hospital between April 1996 and July 1998. The lymphatic mapping has been performed through the preoperative intradermal injection of vital blue dye and technetium-labelled albumin. 319 sentinel lymph nodes were harvested and the 11.3% (15% of patients) were positive for melanoma metastases. No metastases were found in melanomas < or = 1 mm. The percentage of positive sentinel lymph nodes in patients with melanomas > 1 mm in thickness was 16.3% (22% of patients). In 5 cases (2.5%) nodal nevi were found, 1 of which was associated with micrometastasis. All 30 patients with positive sentinel lymph nodes underwent regional lymph node dissection and 555 lymph nodes were harvested. Melanoma metastases were found in only 7 patients, in 31 lymph nodes. The procedure of SLN detection and biopsy is a feasible surgical approach to melanoma patients. It is extremely useful in finding early metastases and in effective pathologic staging. As a consequence of the very low incidence of metastases in the sentinel lymph nodes of patients with thin melanomas, we suggest the sentinel lymph node mapping should be offered to patients with primary melanomas at least 1 mm in depth.     

Nodal melanocytic nevi in sentinel lymph nodes. Correlation with melanoma-associated cutaneous nevi

Melanocytic nevi occurring in lymph nodes create diagnostic difficulty by mimicking metastases. Few studies describe nodal nevi in sentinel lymph nodes (SLNs) excised for melanoma. We evaluated 72 cases in which patients had undergone SLN biopsy for melanoma. Lymph nodes and cutaneous melanomas were evaluated according to a standard protocol. Nodal nevi were identified in 8 patients (11%). Of these, 6 (75%) had an associated cutaneous nevus (P = .006). Of 21 patients with an associated nevus, 4 (19%) with nodal nevi had a cutaneous nevus with congenital features (P = .01). The incidence of nodal nevus correlated with a Breslow thickness greater than 2.5 mm (P = .02). Nevi were not seen in non-SLNs. Nodal nevi appear more frequently in patients with melanoma-associated cutaneous nevi, particularly if congenital features are present. The increased frequency of nodal nevi in SLNs relative to non-SLNs suggests an etiology of mechanical transport of nevus cells.     

Sentinel lymph node investigation in melanoma: detailed analysis of the yield from step sectioning and immunohistochemistry

AIMS: To evaluate in detail the extent to which step sectioning and immunohistochemical examination of sentinel lymph nodes (SLNs) in patients with melanoma reveal additional node positive patients, to arrive at a sensitive yet workable protocol for histopathological SLN examination. METHODS: The study comprised 29 patients with one or more positive SLN after a successful SLN procedure for clinical stage I/II melanoma. SLNs were lamellated into pieces of approximately 0.5 cm in size. One initial haematoxylin and eosin (H&E) stained central cross section was made for each block. When negative, four step ribbons were cut at intervals of 250 microm. One section from each ribbon was stained with H&E, and one was used for immunohistochemistry (IHC). RESULTS: When taking the cumulative total of detected metastases at level 5 as 100%, the percentage of SLN positive patients increased from 79%, 83%, 83%, 90% to 93% in the H&E sections through levels 1-5, and with IHC these values were 83%, 86%, 90%, 97%, and 100%, respectively. One of six patients in whom metastases were detected at levels 2-5 only had metastases in the subsequent additional lymph node dissection. CONCLUSIONS: Multiple level sectioning of SLNs (five levels at 250 microm intervals) and the use of IHC detects additional metastases up to the last level in melanoma SLNs. Although more levels of sectioning might increase the yield even further, this protocol ensures a reasonable workload for the pathologist with an acceptable sensitivity when compared with the published literature.     

Processing of sentinel lymph nodes for detection of metastatic melanoma

Within the last years, evaluation of sentinel lymph nodes (SLN) has become the most popular method of early staging of several malignancies, including breast carcinoma and melanoma. Because SLN are reportedly the lymph nodes most likely to contain metastatic deposits, identification of such nodes allows pathologists to examine the tissue in a much more intense manner than with the usual lymphadenectomy specimens containing multiple lymph nodes. However, there is not a universally accepted standard protocol for pathologic processing of the SLN. Initially, the most popular protocols called for bisection of the SLN and examination of serial sections, with or without routinely performed immunohistochemistry. Lately, other protocols have been proposed to try to simplify the histologic analysis while providing at least equivalent results. Here we review the different protocols used for the evaluation of SLN and describe the protocol currently in use at M. D. Anderson Cancer Center (Houston, TX).     

Specificity of tyrosinase and HMB45 PCR in the detection of melanoma metastases in sentinel lymph node biopsies

AIMS: Sentinel lymph node biopsy is an increasingly established procedure in the primary staging of high-risk melanoma patients. However, the laboratory evaluation of sentinel lymph node biopsies is a matter of controversy. The aim of this study was to determine the specificity of polymerase chain reaction (PCR) techniques for the evaluation of lymph nodes with regard to melanoma metastases in comparison with histology and immunohistology. METHODS AND RESULTS: Sentinel lymph nodes (n = 41) from 29 melanoma patients and 29 lymph nodes from 27 patients without melanoma were analysed by histology (H&E) and immunohistology (Melan A, HMB45). cDNA of these lymph nodes was subjected to LightCycler PCR amplification using primers specific for tyrosinase and HMB45. Two melanoma sentinel lymph nodes contained naevus cells by histology and immunohistology and were therefore excluded from further evaluation. Eight (20.5%) of the remaining 39 melanoma sentinel lymph nodes were positive by histology and immunohistology and tyrosinase PCR, 15.4% (6/39) were positive only by tyrosinase PCR, 2.6% (1/39) were positive only by histology and immunohistology. HMB45 PCR revealed positive results in 7.7% (3/39) sentinel lymph nodes, which were also positive by tyrosinase PCR and histology and immunohistology. Of non-melanoma lymph nodes 13.8% (4/29) and 14.8% (4/27) of non-melanoma patients were positive by tyrosinase PCR but negative by histology and immunohistology and HMB45 PCR. Thus, tyrosinase PCR had a specificity of only 85.2%. CONCLUSIONS: The specificity of tyrosinase PCR and the sensitivity of HMB45 PCR are too low to recommend these PCR examinations for the guidance of therapy, in particular complete regional lymph node dissection.     

Detection of micrometastases in bone marrow and sentinel lymph nodes of breast cancer patients

Objective: To study the sensitivity and clinical significance of HE-staining,IHC and RT-PCR in detecting breast cancer micrometastases in bone marrow and sentinel lymph nodes (SLNs). Methods:After general anesthesia, all patients underwent bone marrow puncture and sentinel lymph node biopsy (SLNB) by 1% isosulfan blue, and then HE-staining,IHC and RT-PCR were used to detect micrometastases. Results:Of 62 patients with breast cancer whose axillary lymph nodes showed negative HE-staining results, 15 cases presented with positive RT-PCR and 9 cases showed positive IHC results positive in bone marrow micrometastases detection. PT-PCR and IHC showed good uniformity(kappa=0.6945)and there was significant difference in detective rate between these two methods (χ2=4.1667,P=0.0412). In SLN samples, 13 showed positive RT-PCR results, while 7 showed positive IHC results. PT-PCR and IHC showed good uniformity (kappa=0.6483)and significant difference was also found in detective rate between these two methods (χ2=4.1667,P=0.0412). Both bone marrow and SLN samples were RT-PCR positive in 3 cases,which indicated that bone marrow micrometastases did not always accompany SLN micrometastases(χ2=0.067,P=0.796). Conclusion: Even if no axillary lymph node involvement or distant metastases are present in routine preoperative examination, micrometastases can still be detected in bone marrow or SLNs. Because the bone marrow micrometastases and axillary node micrometastses are not present simultaneously, combination test of multiple indicators will detect micrometastases more accurately.     

Clinical aspects of sentinel lymph node biopsy in melanoma

Sentinel lymph node (SLN) biopsy has evolved over the years to become one of the most useful tools in the treatment of melanoma. Large, multi-institutional studies have confirmed that in experienced hands it is an accurate, reliable technique to identify tumor in the draining lymph nodes. The complications of the procedure are low, and it is generally well tolerated. The presence of melanoma in the sentinel lymph node is the most important predictive factor in patients with intermediate thickness melanomas. In patients with thin melanomas (<1 mm), the incidence of a positive SLN is low. The choice to perform a SLN biopsy in these patients must be weighed with risk factors such as Clark level, ulceration, sex and mitotic rate. In patients with thick melanomas (>4 mm), most studies have supported the prognostic value of SLN status. Patients with desmoplastic melanomas have a high risk of local recurrence, but low risk of SLN metastasis if the pathology demonstrates a pure desmoplastic form. Younger patients have a higher incidence of positive lymph nodes, yet an overall more favorable prognosis compared to older patients. Patients should understand that this procedure remains primarily a staging tool, as large prospective randomized trials have not demonstrated an overall survival benefit.     

Iatrogenically false positive sentinel lymph nodes in breast cancer: Methods of recognition and evaluation

With the introduction of sentinel lymph node (SLN) biopsy as a standard procedure for staging clinically node negative breast cancer patients, meticulous pathologic evaluation of SLNs by serial sections and/or immunohistochemistry for cytokeratins has become commonplace in order to detect small volume metastases (isolated tumor cells and micrometastases). This practice has also brought to the fore the concept of iatrogenically false positive sentinel nodes secondary to epithelial displacement produced largely by preoperative needling procedures. While this concept is well described in the clinical and pathologic literature, it is, in our experience, still under-recognized, with such lymph nodes frequently incorrectly diagnosed as harboring true metastases, possibly resulting in unwarranted further surgery and/or chemotherapy. This review discusses the concept of displaced epithelium in the histologic evaluation of breast surgical specimens and provides a stepwise approach to the correct identification of iatrogenically transported displaced epithelial cells in sentinel lymph nodes.     

Intraoperative sentinel lymph node evaluation in patients with node-positive breast cancer status post neoadjuvant systemic therapy - An institutional experience

Recent studies have shown the feasibility and utility of sentinel lymph node (SLN) biopsy in patients with biopsy proven node-positive breast cancer after neoadjuvant chemotherapy. We reviewed our experience in intraoperative SLN evaluation in such cases and its effect on axillary management. A retrospective analysis of breast cancer patients (2015–2018) with a biopsy-proven positive axillary lymph node, who received neoadjuvant systemic therapy and underwent intraoperative SLN assessment was performed. Intraoperative SLN assessment results were compared with final pathology. Its accuracy and effect on axillary management is summarized. We identified 106 patients with positive axillary lymph node and neoadjuvant systemic therapy between the ages of 28 and 75 years who had SLN biopsy and lumpectomy (33) or mastectomy (73). Three or more SLNs were identified in 91 cases (86 %). The previously biopsied lymph node was identified as one of the sentinel lymph nodes in 93 cases (88 %). There is a high concordance rate between frozen section diagnosis and final diagnosis on sentinel lymph nodes. No false positive case and seven false negative frozen section diagnosis cases (diagnosed as negative on frozen section and positive on permanent sections) were identified. False-negative frozen section diagnosis correlated with low-volume nodal disease and obscuring tumor bed changes. Almost half of the positive lymph nodes were converted to negative after neoadjuvant chemotherapy. SLN biopsy with intraoperative frozen section evaluation after neoadjuvant systemic therapy in node-positive patients is an effective way to minimize axillary surgery.     

Micromorphometric features of positive sentinel lymph nodes predict involvement of nonsentinel nodes in patients with melanoma

The aim of the present study was to determine whether micromorphometric features of positive sentinel lymph nodes (SLNs) from patients with melanoma are useful for predicting further nodal involvement in completion lymph node dissection (CLND) specimens. Of 986 patients with melanoma undergoing SLN biopsy between March 1992 and February 2001, 175 (17.7%) had at least 1 positive SLN and 140 had subsequent CLND specimens available for review. Further nodal involvement in CLND specimens was present in 24 (17.1%) of 140 patients. Of 8 micromorphometric features of the SLNs that were assessed, the presence of metastases in CLND specimens was correlated significantly with a tumor penetrative depth (maximum distance of melanoma cells from the inner margin of the SLN capsule) of more than 2 mm (P < .05), a deposit size of more than 10 mm2 (P < .01), the presence of melanoma cells in perinodal lymphatic vessels (P < .01), and the effacement of nodal architecture by metastatic melanoma cells (P < .05). Our results indicate that some morphologic features of melanoma metastases in SLNs predict the likelihood of further nodal involvement in CLND specimens.     

Molecular lymphatic mapping of the sentinel lymph node

Intraoperative lymphatic mapping to identify the sentinel lymph node (SLN) has significantly changed the management of regional lymph node basin of patients with various types of solid tumors such as melanoma and breast cancer. The procedure has improved the diagnosis of micrometastasis in the regional tumor-draining lymph nodes by providing a focused histopathological assessment of select lymph nodes most likely to harbor occult disease. Blue dye and/or radioisotopes are efficient mapping agents but the lack of accurate methods to quantify their presence and the potential for dissolution and decay, respectively, throughout time limit their role as reliable markers for identifying a sentinel node from additional secondary lymph nodes that may be either blue and/or radioactive to some degree. A consistently durable marker is needed that can be introduced during surgery and successfully quantitated among tumor-draining lymph nodes to permit a more accurate assessment of hierarchical organization. This may be of particular importance in retrospective analysis of archival tissues as there are no inherent markers to denote the SLN from successive echelon nodes. A procedure of molecular lymphatic mapping (MLM) was developed in a rat model to label the SLN preoperatively with rice gene DNA containing plasmid or linear rice DNA fragment (rDNA). The MLM efficiency was demonstrated by polymerase chain reaction (PCR) detection of the molecular marker in both frozen and paraffin-embedded SLN; 1.25 micro g of rDNA injected with blue dye could be reproducibly detected by PCR. The MLM procedure was validated in a rat breast tumor model with lymph node metastasis. The procedure was successful in permanently labeling and identifying by PCR both frozen and paraffin-embedded SLN. MLM in conjunction with a conventional mapping agent can be used as a valuable asset for molecular assessment of the SLN and retrospective analysis of paraffin-embedded specimens.     

Sentinel lymph node biopsy in melanoma: beyond histologic factors

Sentinel lymph node (SLN) biopsy should be performed with the technical expertise required to correctly identify the sentinel node, in the context of understanding both the likelihood of positivity in a given patient and the prognostic significance of a positive or negative result. National Comprehensive Cancer Network guidelines recommend SLN biopsy for all cutaneous melanoma patients with primary tumor thickness greater than 1 mm and in select patients with thickness between 0.8 and 1 mm, yet admit a lack of consistent clarity in its utility for prognosis and therapeutic value in tumors?<?1 mm and leave the decision for undergoing the procedure up to the patient and treating physician. Recent studies have evaluated specific patient populations, tumor histopathologic characteristics, and gene expression profiling and their use in predicting SLN positivity. These data have given insight into improving the physician’s ability to potentially predict SLN positivity, shedding light on if and when omission of SLN biopsy in specific patients based on clinicopathological characteristics might be appropriate. This review provides discussion and insight into these recent advancements.

     

Interval sentinel lymph nodes in melanoma: a digital pathology analysis of Ki67 expression and microvascular density

The presence of interval sentinel lymph nodes in melanoma is documented in several studies, but controversies still exist about the management of these lymph nodes. In this study, an immunohistochemical evaluation of tumor cell proliferation and neo-angiogenesis has been performed with the aim of establishing a correlation between these two parameters between positive and negative interval sentinel lymph nodes. This retrospective study reviewed data of 23 patients diagnosed with melanoma. Bioptic specimens of interval sentinel lymph node were retrieved, and immunohistochemical reactions on tissue sections were performed using Ki67 as a marker of proliferation and CD31 as a blood vessel marker for the study of angiogenesis. The entire stained tissue sections for each case were digitized using Aperio Scanscope Cs whole-slide scanning platform and stored as high-resolution images. Image analysis was carried out on three selected fields of equal area using IHC Nuclear and Microvessel analysis algorithms to determine positive Ki67 nuclei and vessel number. Patients were divided into positive and negative interval sentinel lymph node groups, and the positive interval sentinel lymph node group was further divided into interval positive with micrometastasis and interval positive with macrometastasis subgroups. The analysis revealed a significant difference between positive and negative interval sentinel lymph nodes in the percentage of Ki67-positive nuclei and mean vessel number suggestive of an increased cellular proliferation and angiogenesis in positive interval sentinel lymph nodes. Further analysis in the interval positive lymph node group showed a significant difference between micro- and macrometastasis subgroups in the percentage of Ki67-positive nuclei and mean vessel number. Percentage of Ki67-positive nuclei was increased in the macrometastasis subgroup, while mean vessel number was increased in the micrometastasis subgroup. The results of this study suggest that the correlation between tumor cell proliferation and neo-angiogenesis in interval sentinel lymph nodes in melanoma could be used as a good predictive marker to distinguish interval positive sentinel lymph nodes with micrometastasis from interval positive lymph nodes with macrometastasis subgroups.