Double Immunohistochemical Labelling of PRAME and Melan A in Slow Mohs Biopsy Margin Assessment of Lentigo Maligna and Lentigo Maligna Melanoma.

Introduction: Lentigo maligna (LM) and lentigo maligna melanoma (LMM) predominantly affect the head and neck areas in elderly patients, presenting as challenging ill-defined pigmented lesions with indistinct borders. Surgical margin determination for complete removal remains intricate due to these characteristics. Morphological examination of surgical margins is the key form of determining successful treatment in LM/LMM and underpin the greater margin control provided through the Slow Mohs micrographic surgery (SMMS) approach. Recent assessments have explored the use of immunohistochemistry (IHC) markers, such as Preferentially Expressed Antigen in Melanoma (PRAME), to aid in LM/LMM and margin evaluation, leveraging the selectivity of PRAME labelling in malignant melanocytic neoplasms. Methods: A Novel double-labelling (DL) method incorporating both PRAME and MelanA IHC was employed to further maximise the clinical applicability of PRAME in the assessment of LM/LMM in SMMS biopsies. The evaluation involved 51 samples, comparing the results of the novel DL with respective single-labelling (SL) IHC slides. Results: The findings demonstrated a significant agreement of 96.1% between the DL method and SL slides across the tested samples. The benchmark PRAME SL exhibited a sensitivity of 91.3% in the SMMS specimens and 67.9% in histologically confirmed positive margins. Discussion: This study highlights the utility of PRAME IHC and by extension PRAME DL as an adjunctive tool in the assessment of melanocytic tumours within staged excision margins in SMMS samples.

PRAME immunohistochemistry as an adjunct for diagnosis and histological margin assessment in lentigo maligna.

AIMS: Lentigo maligna (LM), the most common type of melanoma in situ, is a diagnostically challenging lesion for pathologists due to abundant background melanocytic hyperplasia in sun-damaged skin. Currently, no laboratory methods reliably distinguish benign from malignant melanocytes. However, preferentially expressed antigen in melanoma (PRAME) has shown promise in this regard, and could potentially be applied to diagnosis and margin assessment in difficult cases of LM. METHODS AND RESULTS: Ninety-six cases with a diagnosis of LM (n = 77) or no residual LM (n = 19) following initial biopsy were identified and stained with an antibody directed towards PRAME. Immunohistochemistry (IHC) was scored as positive or negative, and measurement of histological margins by PRAME was performed and compared to the measurement of histological margins using conventional methods [haematoxylin and eosin (H&E) and/or sex-determining region Y-box 10 (SOX10) and/or Melan-A]. Of cases with LM, 93.5% (72 of 77) were PRAME+ and 94.7% (18 of 19) of cases with no residual LM were PRAME- . Of the 35 cases with no margin involvement by PRAME or conventional assessment, 14 cases (40.0%) had no difference in measurement, 17 (48.6%) had a difference of 1 mm or less and four (11.4%) differed by between 1 and 3.5 mm. There was a high correlation between margin assessment methods (r = 0.97, P < 0.0001). CONCLUSIONS: PRAME IHC is a sensitive (93.5%) and specific (94.7%) method for diagnosing LM on biopsy and excision, and measurement of histological margins by PRAME shows a high correlation with conventional methods for margin assessment. Furthermore, the nuclear expression of PRAME makes it a good target for use in dual-colour IHC stains.

Immunohistochemistry utilization in the diagnosis of melanoma.

BACKGROUND: The use of immunohistochemical (IHC) stains in dermatopathology is commonplace; however, little is known regarding utilization trends in melanoma diagnosis. Current Medicare local coverage determinations (LCDs) state that most pigmented lesions, including melanoma, can be diagnosed using H&E alone. METHODS: Histopathology reports for all biopsy-proven melanomas excised between January 1, 2017 and June 30, 2018, at a single dermatology clinic, were identified with the following parameters abstracted: laboratory/dermatopathologist rendering the diagnosis, whether IHC was performed, type/number of stains utilized, presence/depth of invasion, and melanoma subtype. The association of characteristics with IHC utilization was evaluated using χ2 test for categorical variables. RESULTS: Three hundred and fifty six eligible melanomas were identified. IHC was employed in 228 (64%) of the diagnoses. Invasive melanoma was diagnosed in 199 cases (55.9%) while 157 (44.1%) were identified as melanoma in situ (MIS). Of the 228 that utilized IHC, 117 were performed on invasive melanoma (58.8%) and 111 were performed on MIS (70.7%). CONCLUSION: Our findings suggest a higher IHC usage for the diagnosis of melanoma than previously reported. Existing LCDs regarding IHC utilization in melanoma do not reflect the current state of practice. Further investigation regarding IHC utilization and the development of appropriate-use criteria for melanoma IHC is necessary.

Mohs micrographic surgery for melanoma: A prospective multicenter study.

BACKGROUND: Single-institution studies show that frozen section Mohs micrographic surgery (MMS) is an effective treatment modality for cutaneous melanoma, but no multi-institutional studies have been published. OBJECTIVE: To characterize the use of MMS in the treatment of melanoma at 3 academic and 8 private practices throughout the United States, to recommend excision margins when 100% histologic margin evaluation is not used, and to compare actual costs of tumor removal with MMS vs standard surgical excision. METHODS: Prospective, multicenter, cohort study of 562 melanomas treated with MMS with melanoma antigen recognized by T cells 1 immunostaining. RESULTS: Primary trunk and extremity melanomas (noninvasive and invasive melanoma) achieved histologically negative margins in 97% of tumors with 10-mm margins, whereas 12-mm margins were necessary to achieve histologically negative margins in 97% of head and neck melanomas. Overall average cost per tumor treated was $1328.46. LIMITATIONS: Nonrandomized and noncontrolled study. CONCLUSIONS: MMS with melanoma antigen recognized by T cells 1 immunostaining safely provides tissue conservation and same-day reconstruction of histologically verified tumor-free margins in a convenient, single-day procedure. When comprehensive margin evaluation is not used, initial surgical margins of at least 10 mm for primary trunk/extremity and 12 mm for head/neck melanomas should be used to achieve histologically negative margins 97% of the time.

Utility of Multistep Protocols in the Analysis of Sentinel Lymph Nodes in Cutaneous Melanoma: An Assessment of 194 Cases.

CONTEXT.­: Currently, no universal protocol exists for the assessment of sentinel lymph nodes (SLNs) in cutaneous melanoma. Many institutions use a multistep approach with multiple hematoxylin-eosin (H&E) and immunohistochemical stains. However, this can be a costly and time- and resource-consuming task. OBJECTIVE.­: To assess the utility for multistep protocols in the analysis of melanoma SLNs by specifically evaluating the Calgary Laboratory Services (CLS) protocol (which consists of 3 H&E slides and 1 S100 protein, 1 HMB-45, and 1 Melan-A slide per melanoma SLN block) and to develop a more streamlined protocol. DESIGN.­: Histologic slides from SLN resections from 194 patients with diagnosed cutaneous melanoma were submitted to the CLS dermatopathology group. Tissue blocks were processed according to the CLS SLN protocol. The slides were re-reviewed to determine whether or not metastatic melanoma was identified microscopically at each step of the protocol. Using SPSS software, a decision tree was then created to determine which step most accurately reflected the true diagnosis. RESULTS.­: We found with Melan-A immunostain that 337 of 337 negative SLNs (100%) were correctly diagnosed as negative and 55 of 56 positive nodes (98.2%) were correctly diagnosed as positive. With the addition of an H&E level, 393 of 393 SLNs (100%) were accurately diagnosed. CONCLUSIONS.­: We recommend routine melanoma SLN evaluation protocols be limited to 2 slides: 1 H&E stain and 1 Melan-A stain. This protocol is both time- and cost-efficient and yields high diagnostic accuracy.

Low recurrence rates for in situ and invasive melanomas using Mohs micrographic surgery with melanoma antigen recognized by T cells 1 (MART-1) immunostaining: tissue processing methodology to optimize pathologic staging and margin assessment.

BACKGROUND: Various methods of tissue processing have been used to treat melanoma with Mohs micrographic surgery (MMS). OBJECTIVE: We describe a method of treating melanoma with MMS that combines breadloaf frozen sectioning of the central debulking excision with complete peripheral and deep microscopic margin evaluation, allowing detection of upstaging and comprehensive pathologic margin assessment before reconstruction. METHODS: We conducted a retrospective cohort study evaluating for local recurrence and upstaging in 614 invasive or in situ melanomas in 577 patients treated with this MMS tissue processing methodology using frozen sections with melanoma antigen recognized by T cells 1 (MART-1) immunostaining. Follow-up was available in 597 melanomas in 563 patients. RESULTS: Local recurrence was identified in 0.34% (2/597) lesions with a mean follow-up time of 1026 days (2.8 years). Upstaging occurred in 34 of 614 lesions (5.5%), of which 97% (33/34) were detected by the Mohs surgeon before reconstruction. LIMITATIONS: Limitations include retrospective study, intermediate follow-up time, and that the recurrence status of 39.6% of patients was self-reported. CONCLUSION: Treating melanoma with MMS that combines breadloaf sectioning of the central debulking excision with complete peripheral and deep microscopic margin evaluation permits identification of upstaging and consideration of sentinel lymph node biopsy before definitive reconstruction and achieves low local recurrence rates compared with conventional excision.

Assessment of melanoma-initiating cell markers and conventional parameters in sentinel lymph nodes of malignant melanoma.

Sentinel lymph node (SLN) biopsies have widely been used for the detection of occult LN metastasis of malignant melanoma (MM). In addition to conventional biomarkers, we assessed the diagnostic and prognostic significance of melanoma-initiating cell (MIC) markers in SLNs of MM. We examined the expressions of gp100, MART-1 and tyrosinase mRNA for routine diagnosis and those of ABCB5, CD133, nestin, KDM5B, NGFR and RANK mRNA as MIC markers. The presence of micrometastasis was confirmed immunohistochemically using antibodies to S-100, HMB-45, MART-1, and tyrosinase. Discordance between immunohistochemical and molecular data was observed in 14 of 70 (20.0%) patients, among whom five (7.1%) were positive for only molecular markers;two of these five patients tested positive for micrometastasis by repeated immunohistochemical stainings. The quantitative expression levels of gp100, MART-1, and tyrosinase mRNA were significantly higher in the metastatic LNs;the cut-off values remain to be elucidated. ABCB5 mRNA expression was detected more frequently in the metastatic SLNs (p＜0.05) and in the group of patients with recurrence. To make a definite diagnosis of metastasis, we still need a combination of immunohistochemical and molecular probes. ABCB5 might be a suitable molecular marker for the detection of melanoma-initiating cells in SLNs.

Expression of soluble adenylyl cyclase in lentigo maligna: use of immunohistochemistry with anti-soluble adenylyl cyclase antibody (R21) in diagnosis of lentigo maligna and assessment of margins.

CONTEXT: Soluble adenylyl cyclase (sAC) is an enzyme that generates cyclic adenosine monophosphate, a signaling molecule involved in regulating melanocyte functions. R21, a mouse monoclonal antibody against sAC, shows a striking pan-nuclear staining in lentigo maligna, indicating possible utility for diagnosis and margin assessment. OBJECTIVE: To evaluate R21 in the diagnosis and evaluation of margins in lentigo maligna. DESIGN: Thirty one re-excision specimens for lentigo maligna were evaluated for R21 expression using previously published protocol. In addition, 153 cases including 41 lentigo malignas, 30 non-lentigo maligna-type melanomas, 38 lentigos, and 44 nevi were evaluated using a modified stringent protocol to eliminate all nonmelanocyte staining. RESULTS: The sensitivity of nuclear staining with R21 in lentigo maligna was 87.8%. Nuclear expression of sAC was observed in 40% of other melanomas and 2.3% of benign nevi. R21 did not stain nuclei of resting melanocytes but was observed in 28.9% of melanocytic hyperplasias. These cases were easily distinguished from lentigo maligna in routine sections. R21 staining facilitated extent of the lesion in resection margins. In cases examined under the less stringent conditions, interpretation was facilitated by comparing R21 and Mart1/Melan A staining. Greater than 9 pan-nuclear staining melanocytes within one high-power field along with a pan-nuclear sAC/Melan A ratio greater than 0.5 was consistent with a positive margin whereas 5 or less pan-nuclear staining melanocytes along with a sAC/Melan A ratio of less than 0.3 constituted a negative margin. CONCLUSION: R21 is a useful diagnostic adjunct in the diagnosis and evaluation of margins in re-excision specimens in lentigo maligna.

Flow cytometry assessment of residual melanoma cells in tumor-infiltrating lymphocyte cultures.

Adoptive transfer of tumor-infiltrating lymphocytes (TIL) is in development for the treatment of metastatic melanoma. In phase II clinical trials, patients with metastatic melanoma that received TIL after preconditioning had a 50-70% clinical response rate. The current approach to generate TIL is to culture melanoma enzyme digests in the presence of IL-2 for a 10- to 20-day period followed by 2 weeks of rapid expansion (REP). Prior to administration, cell therapies are characterized and tested for purity. TIL are characterized by CD3 surface marker expression, and purity is assessed by the amount of tumor remaining in culture. Evaluating TIL purity has traditionally been done by immunohistochemistry, which is often considered semiquantitative. To generate a quantitative assay, we used multiparameter flow cytometry to evaluate the presence of viable tumor cells by staining TIL populations with a viability dye and an antibody cocktail that detects intracellular tumor-antigens gp100, Mart-1, tyrosinase, S100, and surface tumor-antigen melanoma chondroitin sulfate proteoglycan (MCSP), and CD3 on T cells. Tumors were identified by gating on the viable CD3(-) population. Antigens in tumors were initially optimized with individual antibodies using both immunohistochemistry and flow cytometry. When eight different tumor cell lines were spiked into an activated T cell culture, flow cytometry was able to distinguish lymphocytes from tumors in all samples tested. Most importantly, the assay was able to detect melanoma cells in all enzyme digests (9/9) from patient samples. After IL-2-induced TIL expansion, there was a significant decrease in tumor cells; tumor cells were detected in only 2 of 12 samples. In eight IL-2-induced TIL samples that were further expanded in REP, no tumor cells were detected. We have demonstrated that flow cytometry is an alternative to immunohistochemistry for defining the purity of a TIL population.

Immunohistochemical dual staining as an adjunct in assessment of mitotic activity in melanoma.

BACKGROUND: The mitotic rate was introduced as a major prognostic criterion for the staging of thin (≤1.0 mm) melanoma by the 2009 American Joint Committee on Cancer Staging and Classification (seventh edition). The detection of a single mitotic figure changes the tumor stage in thin melanoma. We sought to address the value of a dual staining to facilitate the determination of the mitotic rate and to assign the mitotic activity to melanocytes. METHODS: The mitotic rate of melanoma cells was determined by dual phosphohistone-H3 (PHH3)/Melan-A immunohistochemistry. Results were compared with PHH3 staining alone and conventional hematoxylin and eosin (H&E)-stained slides of 15 melanomas with a tumor thickness <1.0 mm. RESULTS: PHH3 staining clearly labeled cells in the mitotic cell cycle. The mitotic rate in the PHH3/Melan-A dual stain was equal to that derived by H&E staining. Time required for counting mitotic figures was significantly reduced. CONCLUSIONS: The evaluation of mitotic rate with an immunohistochemical dual stain is faster (mean 63.0%) and more reliable than evaluation by routine H&E staining alone. Dual staining immunohistochemistry may be a useful additional tool to standardize the determination of mitotic rate and may be helpful in evaluation of challenging cases.

Assessment of prognostic circulating tumor cells in a phase III trial of adjuvant immunotherapy after complete resection of stage IV melanoma.

OBJECTIVE: To verify circulating tumor cell (CTC) prognostic utility in stage IV resected melanoma patients in a prospective international phase III clinical trial. BACKGROUND: Our studies of melanoma patients in phase II clinical trials demonstrated prognostic significance for CTCs in patients with AJCC stage IV melanoma. CTCs were assessed to determine prognostic utility in follow-up of disease-free stage IV patients pre- and during treatment. METHODS: After complete metastasectomy, patients were prospectively enrolled in a randomized trial of adjuvant therapy with a whole-cell melanoma vaccine, Canvaxin, plus Bacille Calmette-Guerin (BCG) versus placebo plus BCG. Blood specimens obtained pretreatment (n = 244) and during treatment (n = 214) were evaluated by quantitative real-time reverse-transcriptase polymerase chain reaction (qPCR) for expression of MART-1, MAGE-A3, and PAX3 mRNA biomarkers. Univariate and multivariate Cox analyses examined CTC biomarker expression with respect to clinicopathological variables. RESULTS: CTC biomarker(s) (≥ 1) was detected in 54% of patients pretreatment and in 86% of patients over the first 3 months. With a median follow-up of 21.9 months, 71% of patients recurred and 48% expired. CTC levels were not associated with known prognostic factors or treatment arm. In multivariate analysis, pretreatment CTC (> 0 vs. 0 biomarker) status was significantly associated with disease-free survival (DFS; HR 1.64, P = 0.002) and overall survival (OS; HR 1.53, P = 0.028). Serial CTC (>0 vs. 0 biomarker) status was also significantly associated with DFS (HR 1.91, P = 0.02) and OS (HR 2.57, P = 0.012). CONCLUSION: CTC assessment can provide prognostic discrimination before and during adjuvant treatment for resected stage IV melanoma patients.

Direct evidence on the immune-mediated spontaneous regression of human cancer: an incentive for pharmaceutical companies to develop a novel anti-cancer vaccine.

To develop an effective pharmaceutical treatment for a disease, we need to fully understand the biological behavior of that disease, especially when dealing with cancer. The current available treatment for cancer may help in lessening the burden of the disease or, on certain occasions, in increasing the survival of the patient. However, a total eradication of cancer remains the researchers' hope. Some of the discoveries in the field of medicine relied on observations of natural events. Among these events is the spontaneous regression of cancer. It has been argued that such regression could be immunologically-mediated, but no direct evidence has been shown to support such an argument. We, hereby, provide compelling evidence that spontaneous cancer regression in humans is immunologically-mediated, hoping that the results from this study would stimulate the pharmaceutical industry to focus more on cancer vaccine immunotherapy. Our results showed that patients with >3 primary melanomas (very rare group among cancer patients) develop significant histopathological spontaneous regression of further melanomas that they could acquire during their life (P=0.0080) as compared to patients with single primary melanoma where the phenomenon of spontaneous regression is absent or minimal. It seems that such regression resulted from the repeated exposure to the tumor which mimics a self-immunization process. Analysis of the regressing tumors revealed heavy infiltration by T lymphocytes as compared to non-regressing tumors (P<0.0001), the predominant of which were T cytotoxic rather than T helper. Mature dendritic cells were also found in significant number (P<0.0001) in the regressing tumors as compared to the non regressing ones, which demonstrate an active involvement of the different arms of the immune system in the multiple primary melanoma patients in the process of tumor regression. Also, MHC expression was significantly higher in the regressing versus the non-regressing tumors (P <0.0001), which reflects a proper tumor antigen expression. Associated with tumor regression was also loss of the melanoma common tumor antigen Melan A/ MART-1 in the multiple primary melanoma patients as compared to the single primary ones (P=0.0041). Furthermore, loss of Melan A/ MART-1 in the regressing tumors significantly correlated with the presence of Melan A/ MART-1-specific CTLs in the peripheral blood of these patients (P=0.03), which adds to the evidence that the phenomenon of regression seen in these patients was immunologically-mediated and tumor-specific. Such correlation was also seen in another rare group of melanoma patients, namely those with occult primary melanoma. The lesson that we could learn from nature in this study is that inducing cancer regression using the different arms of the immune system is possible. Also, developing a novel cancer vaccine is not out of reach.

Pathologic assessment of melanoma sentinel nodes: a role for molecular analysis using quantitative real-time reverse transcription-PCR for MART-1 and tyrosinase messenger RNA.

PURPOSE: Molecular analysis of melanoma sentinel nodes (SN) is sensitive, but poorly specific because metastases cannot be distinguished from benign nevus inclusions (BNI). We investigated whether quantitative reverse transcription-PCR (RT-PCR) detection of MART-1 and tyrosinase mRNAs could improve this specificity and contribute to SN assessment. EXPERIMENTAL DESIGN: Two hundred twenty SNs from 95 melanoma patients analyzed by extensive immunohistopathology and real-time quantitative RT-PCR. RESULTS: Using histopathology, SNs and patients were allotted to three diagnostic groups: (a) metastasis positive, (b) BNI positive, and (c) melanocyte-free. Median MART-1 and tyrosinase mRNA levels in SNs were significantly different in patients with metastasis compared with patients with BNIs (P < 0.05) and patients without melanocytic lesions (P < 0.001). However, a "gray-zone" was observed where distinction, based on mRNA levels, could not be made between the three groups. For both genes, the highest mRNA level recorded in each RT-PCR-positive patient was positively correlated with Breslow's tumor thickness. For SNs with metastases, tumor burden was significantly correlated to the mRNA level. Using the presence of a MART-1 RT-PCR signal to detect patients with metastases, a sensitivity of 100% and a negative predictive value of 100% were achieved when extensive immunohistology was used as reference. CONCLUSIONS: Quantitative RT-PCR MART-1 and tyrosinase mRNA analysis cannot be used alone for SN diagnosis because of its poor specificity for melanoma metastasis. However, in approximately one third of cases without RT-PCR evidence of MART-1 expression, extensive histopathologic SN investigation is not necessary, thus substantially reducing the cost of SN analysis. The level of melanocyte-associated mRNA is associated with both tumor thickness and tumor burden as measured histopathologically, suggesting that this may be of prognostic value.

Quantitative detection of circulating tumor cells in cutaneous and ocular melanoma and quality assessment by real-time reverse transcriptase-polymerase chain reaction.

PURPOSE: Inconsistent reports on the detection of melanoma cells in peripheral blood by reverse transcriptase-PCR (RT-PCR) have resulted in uncertainty on the prognostic value of circulating melanoma cells. EXPERIMENTAL DESIGN: We developed real-time RT-PCR assays for quantitation of tyrosinase, MelanA/MART1, and gp100 and for porphobilinogen deaminase housekeeping gene. Melanoma tissue (n = 18), peripheral blood samples from healthy donors (n = 21), and patients with cutaneous (n = 122) and uveal (n = 64) melanoma from our institution were analyzed. For quality control, an additional 251 samples from ongoing multicenter studies were compared with in-house samples. RESULTS: Tyrosinase was not detected in healthy donor blood samples. For the two other markers, cutoff values had to be defined to distinct patient samples from controls. Patients with stage IV uveal and cutaneous melanoma expressed all three markers more frequently and at higher levels in peripheral blood as compared with earlier stages. The variation of expression was 4 logs and correlated with tumor load and serum lactate dehydrogenase. In 2 of 3 uveal melanoma patients, detection of circulating tumor cells preceded the development of liver metastases. The diagnostic sensitivity was optimal in blood samples containing >0.1pg/ microl porphobilinogen deaminase (95.7% of in-house samples and 57.4% of multicenter samples). CONCLUSIONS: Real-time RT-PCR is able to quantitatively define the quality of a sample and provides quantitative data for melanoma markers. Disparities in the results of previous studies may be attributable to undetected differences in sample quality. The prognostic relevance of this assay is currently under evaluation in several prospective randomized trials.

Accurate molecular detection of melanoma nodal metastases: an assessment of multimarker assay specificity, sensitivity, and detection rate.

BACKGROUND: The application of lymphoscintigraphy followed by sentinel lymph node (SN) biopsy to patients with primary melanoma has revolutionised the ability to identify accurately, yet conservatively, those patients who harbour occult nodal metastases. The molecular detection of SN micrometastases facilitates the cost effective analysis of the entire SN using multiple markers. Currently, a lack of marker specificity is the main barrier preventing the molecular evaluation of SN tissue from becoming clinically applicable. AIMS: To develop a reproducible multimarker reverse transcription-polymerase chain reaction (RT-PCR) assay, with the emphasis on achieving high specificity for the accurate detection of melanoma metastases in nodal tissue. METHODS: Three pigment cell specific (PCS) markers-tyrosinase, Pmel-17, and MART-1-and one cancer testis antigen (CTA)-MAGE-3-were selected for use in a multimarker RT-PCR assay. The conditions for this assay were optimised. RESULTS: High specificity was achievable for each marker by optimising the PCR cycle number such that unwanted transcripts (that is, illegitimate transcripts and/or specific transcripts from other low abundance nodal cell types) remained undetectable in appropriate controls (normal visceral nodes). Tyrosinase was 100% specific at 40 PCR cycles, MAGE-3 and MART-1 at 35 PCR cycles, and Pmel-17 at 30 PCR cycles. Tyrosinase proved to be the most sensitive marker, detecting 10 melanoma cells in 0.1 g of nodal tissue. CONCLUSIONS: Excellent reproducibility of the entire nodal processing and RT-PCR protocol for the detection of very low numbers of melanoma cells in nodal tissue was shown, although there is a risk of false positives using the PCS markers alone, because of an approximate 4-8.5% incidence rate of nodal nevi in melanoma draining SNs (these nevi being absent in all other normal nodes). MAGE-3 was shown to be the only marker that is not expressed by melanocytes. However, because not all melanomas express MAGE-3, it is recommended that more emphasis should be placed on the development of a panel of CTA markers to ensure a zero false positive rate and to provide optimum detection.

Quantitative RT-PCR assessment of melanoma cells in peripheral blood during immunotherapy for metastatic melanoma.

Circulating malignant cells in peripheral blood are thought to be precursors and surrogate markers of distant metastases and hence markers of a poor clinical outcome. In this study, we used the detection of MART-1 and tyrosinase (TYR) mRNA with a quantitative reverse transcription-polymerase chain reaction (RT-PCR) assay to identify circulating melanoma cells. Blood samples were obtained from 35 patients with metastatic melanoma before, during and after treatment with interleukin-2, interferon-alpha and cisplatin. In addition, MART-1 and TYR protein was identified by immunohistochemistry in consecutive biopsies from 15 of the patients. Analysis of three daily blood samples for 3 days demonstrated that four out of 11 patients examined were negative for both markers on all occasions, and two patients were positive for both markers on all occasions but one. The remaining five patients showed sporadic low positive results for one or the other of the two markers. By comparing the immunohistochemistry results from consecutive biopsies with the RT-PCR results, we demonstrated that patients with MART-1 and TYR protein in their tumour cells had circulating MART-1 and TYR mRNA in 77% and 54% of the cases, respectively. During treatment, the majority of patients who were positive for MART-1 and TYR mRNA converted to being negative. However, these conversions did not significantly correlate with objective response. The presence of TYR mRNA in one of the first two samples showed a trend towards being an independent prognostic factor for poor survival.

Uveal melanoma cell staining for CD34 and assessment of tumor vascularity.

PURPOSE: Aggressive melanoma cells may express endothelial markers that can be used to calculate microvascular density (MVD). High MVD has been associated with adverse outcome in uveal melanoma. If tumor cells label with endothelial cell markers, then MVD may not accurately reflect a tumor's vascularity. This study was designed to study the influence of melanoma cell labeling by endothelial cell markers on MVD. METHODS: Tissue sections of 200 ciliary body or choroidal melanomas were stained with CD34 alone, and the MVD was calculated by counting discrete foci of CD34 labeling in hot spots, as described previously. From adjacent sections double labeled by fluorescent immunohistochemical stains for S100 protein and CD34, tumor cells labeling with both markers were identified. The relationship between marker coexpression and MVD was tested. Tissue sections were also double labeled for Melan-A and CD34. RESULTS: MVD was found to be associated with death from metastatic melanoma as reported previously. However, colocalization of both Melan-A and S100 protein with CD34 was demonstrated. The labeling of tumor cells by CD34 was associated with an elevated calculation of MVD (P < 0.0001) but not with cell type, mitotic figures, tumor-infiltrating lymphocytes, or PAS-positive patterns. CONCLUSIONS: CD34 may label uveal melanoma cells and may contribute to computation of MVD. Although MVD is prognostically significant in uveal melanoma, this feature is not an exclusive measure of tumor vascularity.