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.

Utility of teledermatopathology for intraoperative margin assessment of melanoma in situ, lentigo maligna type: A 6 year community practice experience.

BACKGROUND: Achieving negative margins for melanoma in situ, lentigo maligna type can be challenging, particularly on cosmetically sensitive areas. OBJECTIVE: To assess the utility of intraoperative frozen section margin assessment using a teledermatopathology system in the treatment of head and neck lentigo maligna. METHODS AND MATERIALS: Over a 6 year period, 96 patients with lentigo maligna had surgical excisions. The margins were assessed intraoperatively with frozen sections prepared in the manner used in Mohs surgery. The surgeon guided the frozen section slides around the margin while a dermatopathologist assessed the margin remotely. RESULTS: In 2/96 (2.1%) cases, the safety margin was positive (frozen sections were false negative). In 1 further case (1%) there was a recurrence of the melanoma 13 months following the excision. CONCLUSION: The described method is effective in treating melanoma in situ, lentigo maligna type with clearance rates similar to previous studies for Mohs surgery.

Confocal Microscopy and Lentigo Maligna: An in vivo Pilot Study for the Assessment of Response to Imiquimod Therapy.

BACKGROUND: Reflectance confocal microscopy (RCM) is a noninvasive technique that provides real-time in vivo images of the epidermal layer. Imiquimod has been recommended as an alternative treatment in lentigo maligna (LM) when surgical excision is not the treatment of choice. In the present study we compare the results of in vivo RCM to the histopathological examination before and after treatment of LM with topical imiquimod. METHODS: Thirty-four patients with confirmed LM were included. Imiquimod 5% was applied until a weeping erosion appeared in the LM-affected skin. Evaluation was performed by clinical examination, dermatoscopy, histopathology and RCM. RESULTS: During the follow-up, 27 of 34 patients (79.42%) demonstrated a total tumor clearance by imiquimod treatment. In the treated area, a significant decrease of atypical cells was detected using RCM (p < 0.0001). Furthermore, a significant positive correlation in the detected atypical cells was shown using confocal microscopy and histology (p = 0.0001, r = 0.7335, respectively). CONCLUSION: In patients not suitable for surgical intervention imiquimod treatment is an appropriate treatment alternative. Thereby, in vivo RCM was demonstrated to be an excellent examining device, which not only allows diagnosis of LM, but also therapy and follow-up examinations. An important benefit of RCM, in contrast to conventional histopathology, is the simple handling with in vivo examination of epidermal skin without any pain for the patient.

The smart approach: feasibility of lentigo maligna superficial margin assessment with hand-held reflectance confocal microscopy technology.

BACKGROUND: Lentigo maligna may be challenging to clear surgically. OBJECTIVE: To evaluate feasibility of using superficial skin cuts as RCM imaging anchors for attaining negative surgical margins in lentigo maligna. METHODS: Included patients presented with lentigo maligna near cosmetically sensitive facial structures. We evaluated, with hand-held-RCM, microscopic clearance of melanoma beyond its dermoscopically detected edges. Evaluated margins were annotated using shallow skin cuts. If a margin was positive at 'first-step' RCM evaluation, we sequentially advanced the margin radially outward at that segment by 2-mm intervals until an RCM-negative margin was identified. Prior to final surgical excision, we placed sutures at the outmost skin cuts to allow comparison of RCM and histopathological margin assessments. Primary outcome measure was histopathological verification that RCM-negative margins were clear of melanoma. RESULTS: The study included 126 first-step margin evaluations in 23 patients, median age 70 years (range: 43-91). Seventeen patients (74%) had primary in-situ melanoma and six (26%) invasive melanoma, mean thickness 0.3 mm (range 0.2-0.4 mm). Six cases (26%) showed complete negative RCM margins on 'first-step', 11 (48%) were negative at 'second-step', and four (17%) at 'third-step'. In two additional cases (9%), margins clearance could not be determined via RCM due to widespread dendritic cells proliferation. The RCM-negative margins in all 21 cases proved clear of melanoma on histopathology. Of the 15 cases that returned at 1-year follow-up, none showed any residual melanoma on dermoscopic and RCM examinations. Interobserver reproducibility showed fair agreement between bedside RCM reader and blinded remote-site reader, with Spearman's rho of 0.48 and Cohen's kappa of 0.43; using bedside reader as reference, the remote reader's sensitivity was 92% and specificity 57% in positive margin detection. CONCLUSIONS: Margin mapping of lentigo maligna with hand-held-RCM, using superficial skin cuts, appears feasible. This approach needs validation by larger studies.

Assessment of changes in lentigo maligna during radiotherapy by in-vivo reflectance confocal microscopy: a pilot study.

BACKGROUND: Radiotherapy is an effective treatment for therapy of lentigo maligna (LM). OBJECTIVES: To investigate the usefulness of in-vivo reflectance confocal microscopy (RCM) in radiotherapy of LM and document the changes within the lesions during treatment. METHODS: A total of six lesions in six patients were investigated by RCM before, during and after radiotherapy. For diagnostic assessment three observers with experience in RCM diagnosis, blinded as to the stage of treatment, assessed the RCM images of each lesion and documented the findings by consensus. RESULTS: Epidermal disarray worsened in three patients during radiotherapy and superficial necrosis was observed in four patients. Large pagetoid round/dendritic cells decreased or even vanished during or after radiotherapy. Dilated vessels and apoptotic cells were seen in all patients during radiotherapy as well as an increase of inflammatory cells in the epidermis and dermis in most of the patients. Dendritic cells with small dendrites were observed during radiotherapy in all patients with an increase in number in three patients. Melanophages appeared in five patients at least once during the examination period. All RCM images were assessed correctly by the three observers. CONCLUSIONS: Reflectance confocal microscopy is a useful method to visualize changes during and after radiotherapy and might also be used for early detection of potential treatment failures. In addition, it might be helpful in planning radiotherapy.

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.

Soluble adenylyl cyclase antibody profile as a diagnostic adjunct in the assessment of pigmented lesions.

OBJECTIVE: To investigate the usefulness of a novel marker for melanocytic proliferations. DESIGN: Using a novel monoclonal antibody against soluble adenylyl cyclase (sAC), various benign and malignant melanocytic proliferations were immunostained. SETTING: Weill Medical College of Cornell University dermatopathology laboratory. MAIN OUTCOME MEASURES: The results were qualitative, not quantifiable. RESULTS: The sAC immunostaining produced distinctive patterns that paralleled melanomagenesis. At one pole of the spectrum were benign nevi, including atypical nevi of special sites and recurrent nevi showing a distinct pattern of dotlike Golgi staining, while at the opposite pole was melanoma, in which many cells demonstrated an intense pannuclear expression pattern, often accompanied by loss of the Golgi expression pattern. Melanomas of lentigo maligna and acral lentiginous subtypes exhibited the most striking pannuclear expression, while nodular melanomas showed the least, although with supervening enhanced diffuse cytoplasmic expression. Loss of the Golgi expression pattern was a feature of malignant melanoma. CONCLUSION: The sAC expression pattern is complex but seems discriminatory, with distinctive and variable staining patterns according to the nature of the lesion biopsied.

Assessment of tumor thickness in melanocytic skin lesions: comparison of optical coherence tomography, 20-MHz ultrasound and histopathology.

BACKGROUND: Accurate assessment of vertical tumor size is important for surgical treatment planning of melanocytic skin lesions. High-frequency ultrasound (HFUS) is frequently used for this purpose, but overestimation of tumor thickness is known as a problem especially in thin melanocytic lesions. Optical coherence tomography (OCT) as a new imaging technique might be a promising alternative. OBJECTIVE: To evaluate the ability of OCT to accurately determine the vertical tumor thickness of melanocytic skin lesions and to compare it with HFUS and histopathology in order to improve surgical planning. METHODS: In this single-center study, 26 melanocytic lesions were imaged by OCT and HFUS. Vertical lesion dimensions of both methods were compared with histopathological measurements. RESULTS: Bland-Altman plots for OCT and histopathology as well as for HFUS and histopathology revealed better agreement for OCT and histopathology concerning tumor thickness measurements. Tumor thickness values for the melanocytic lesions measured by OCT presented a median tumor thickness of 0.31 mm (range 0.10-0.77) compared to a median tumor thickness of 0.25 mm (range 0.06-1.5) measured by histopathology. The median tumor thickness of HFUS was 0.44 mm (range 0.23-1.1). A Spearman correlation procedure including the correlation coefficient (r) showed a stronger relationship between OCT and histopathology (r = 0.734) compared to HFUS and histopathology (r = 0.390). CONCLUSIONS: On the basis of this smaller study cohort, OCT seems to be more exact than HFUS as far as thickness determination of thin melanocytic skin lesions is concerned.

Assessment of the optimal interval for and sensitivity of short-term sequential digital dermoscopy monitoring for the diagnosis of melanoma.

OBJECTIVE: To determine whether 6 weeks could replace 3 months for short-term sequential digital dermoscopy imaging (ST-SDDI) of suspicious melanocytic lesions and determine the proportion of melanomas missed. DESIGN: Consecutive lesions (n = 2602) undergoing ST-SDDI monitored from 1859 patients were included. Half of the patients underwent 6-week monitoring followed by 3-month monitoring (range, 2.5-4.5 months) if changes were not seen. The remainder underwent 3-month monitoring only. Any change during this time led to excision. Lesions unchanged were then followed up over time. SETTING: A tertiary referral institution. MAIN OUTCOME MEASURES: The proportion of changed melanomas (sensitivity) and odds ratios (ORs) for melanoma of changed lesions. RESULTS: Eighty-one melanomas were detected using ST-SDDI (Breslow thickness: median, in situ; maximum, 0.8 mm). Of 39 melanomas detected using ST-SDDI in the 6-week monitored lesions, 27 (69%) were detected at 6 weeks and 12 (31%) at 3 months. The OR for melanoma for a lesion changing at 6 weeks was 19 (95% confidence interval [CI], 10-35), and the overall OR for melanoma for a lesion changing during the short-term monitoring period (6 weeks to 4.5 months) was 47 (95% CI, 23-94). For lesions remaining unchanged at 3 months, 99.2% (1118 of 1127 lesions) were shown to be benign as defined by an unremarkable further follow-up. Seventy-five percent (15 of 20) of the lentigo maligna melanomas, 93% (40 of 43) of other in situ melanomas, and 96% (26 of 27) of the invasive melanomas were detected using ST-SDDI. Conclusion Three months remains the standard interval for ST-SDDI, where the sensitivity for the diagnosis of melanoma for changed (non-lentigo maligna) lesions is high but not 100%.

Prevention campaign against skin cancer.

BACKGROUND: The melanoma incidence has increased over recent decades. Educational campaigns aim to encourage protection from the sun and early detection of melanoma. METHODS: During a campaign in Switzerland, information on risk factors, sun protection and melanoma prevention was distributed. 10987 people completed a questionnaire regarding risk factors, and 2746 people were examined by a dermatologist. RESULTS: Men had a higher risk as assessed by skin type, ultraviolet exposure, family history, number of moles and sunburns during childhood. Changes in moles were reported significantly more often by people with: (1). a positive family history (p < 0.0001); (2). multiple moles (p < 0.0001), and (3). sunburns during childhood (p < 0.0001). A precancerous or cancerous condition was suspected in 16% of individuals examined. CONCLUSION: Early detection of melanoma can be achieved by this type of campaign. Primary prevention is a long-term approach and educational efforts targeting risk groups must be continued.