Morphologic features of melanophages under in vivo reflectance confocal microscopy.

OBJECTIVES: To determine morphologic features of melanophages under in vivo reflectance confocal microscopy (RCM) and to highlight morphologic features that are important in distinguishing melanophages from melanocytes. DESIGN: Consecutive retrospective study. SETTING: Referral center for pigmented lesions. PATIENTS: The study group retrospectively constituted 20 consecutive patients having biopsy-proven lichen planus-like keratoses that dermoscopically and histopathologically showed many melanophages and that had been imaged under RCM before biopsy. MAIN OUTCOME MEASURES: The RCM characteristics of isolated dermal bright cells were scored blinded to dermoscopic features and histopathologic diagnosis. RESULTS: Under RCM, melanophages were significantly smaller than melanocytes (mean [SD] cell diameter, 13.6 [1.6] vs 18.2 [2.9] microm, P = .006). Nuclei (intracellular low-reflectance round-oval structures) were visible in only 16% (29 of 184) of the cells in melanophages vs 57% (28 of 49) of the cells in melanocytes (P < .001). When identified, nuclei were smaller in melanophages than in melanocytes (mean [SD] diameter, 3.2 [1.2] vs 6.4 [0.7] microm, P < .001). Compared with melanocytes, melanophages were significantly more ill defined (76% [140 of 184] vs 18% [9 of 49], P < .001), less round (23% [42 of 184] vs 69% [34 of 49], P < .001), and less dendritic (1% [2 of 184] vs 12% [6 of 49]) (P = .001). CONCLUSION: Observed differences in morphologic features should enable distinction between melanophages and melanocytes under RCM, thereby improving the accuracy of skin lesion diagnosis using this technique.

The impact of in vivo reflectance confocal microscopy on the diagnostic accuracy of lentigo maligna and equivocal pigmented and nonpigmented macules of the face.

Limited studies have reported the in vivo reflectance confocal microscopy (RCM) features of lentigo maligna (LM). A total of 64 RCM features were scored retrospectively and blinded to diagnosis in a consecutive series of RCM sampled, clinically equivocal, macules of the face (n=81 LM, n=203 benign macules (BMs)). In addition to describing RCM diagnostic features for LM (univariate), an algorithm was developed (LM score) to distinguish LM from BM. This comprised two major features each scoring +2 points (nonedged papillae and round large pagetoid cells > 20 microm), and four minor features; three scored +1 point each (three or more atypical cells at the dermoepidermal junction in five 0.5 x 0.5 mm(2) fields, follicular localization of atypical cells, and nucleated cells within the dermal papillae), and one (negative) feature scored -1 point (a broadened honeycomb pattern). A LM score of > or = 2 resulted in a sensitivity of 85% and specificity of 76% for the diagnosis of LM (odds ratio (OR) for LM 18.6; 95% confidence interval: 9.3-37.1). The algorithm was equally effective in the diagnosis of amelanotic lesions and showed good interobserver reproducibility (87%). In a test set of 29 LMs and 44 BMs, the OR for LM was 60.7 (confidence interval: 11.9-309) (93% sensitivity, 82% specificity).

False negative sentinel lymph node biopsies in melanoma may result from deficiencies in nuclear medicine, surgery, or pathology.

OBJECTIVE: To investigate a cohort of melanoma patients with false negative (FN) sentinel node (SN) biopsies (SNBs) to identify the reasons for the FN result. SUMMARY OF BACKGROUND DATA: SNB is a highly efficient staging method in melanoma patients. However, with long-term follow-up FN SNB results of up to 25% have been reported. METHODS: Seventy-four SNs from 33 patients found to have had an FN SNB were analyzed by reviewing the lymphoscintigraphy, surgical data, and histopathology, and by assessing nodal tissue using multimarker real-time quantitative reverse transcription (qRT) polymerase chain reaction, and antimony concentration measurements (as a marker of "true" SN status) using inductively coupled plasma mass spectroscopy. RESULTS: Nine SNs (12%) from 9 patients (27%) had evidence of melanoma on histopathologic review. Twelve SNs (16%) from 10 patients (30%) were qRT(+). Four of these 12 SNs were positive on histopathology review and 8 were negative. Four patients (12%) were upstaged by qRT. Sixteen patients had their SNB histology, lymphoscintigraphy, and surgical data reviewed. Identifiable causes of the FN SNBs were not found after review of all modalities in 4 patients. SNs from all 4 patients had antimony levels indicative of an SN. Of the SNs evaluable by qRT, 1 was qRT(+) and 7 SNs from 2 patients were qRT(-). CONCLUSIONS: An FN SN can occur because of deficiencies in nuclear medicine, surgery, or pathology. qRT can detect "occult" metastatic melanoma in SNs that have been identified as negative by histopathology.

Confirmation of sentinel lymph node identity by analysis of fine-needle biopsy samples using inductively coupled plasma-mass spectrometry.

BACKGROUND: The sentinel lymph node (SLN) biopsy technique is a reliable means of determining the tumor-harboring status of regional lymph nodes in melanoma patients. When technetium 99 m-labeled antimony trisulfide colloid (99 mTc-Sb2S3) particles are used to perform preoperative lymphoscintigraphy for SLN identification, they are retained in the SLN but are absent or present in only tiny amounts in non-SLNs. The present study investigated the potential for a novel means of assessing the accuracy of surgical identification of SLNs. This involved the use of inductively coupled plasma-mass spectrometry (ICP-MS) to analyze antimony concentrations in fine-needle biopsy (FNB) samples from surgically procured lymph nodes. METHODS: A total of 47 FNB samples from surgically excised lymph nodes (32 SLNs and 15 non-SLNs) were collected. The SLNs were localized by preoperative lymphoscintigraphy that used 99 mTc-Sb2S3, blue dye, and gamma probe techniques. The concentrations of antimony were measured in the FNB samples by ICP-MS. RESULTS: The mean and median antimony concentrations (in parts per billion) were .898 and .451 in the SLNs, and .015 and .068 in the non-SLNs, the differences being highly statistically significant (P < .00005). CONCLUSIONS: Our results show that ICP-MS analysis of antimony concentrations in FNB specimens from lymph nodes can accurately confirm the identity of SLNs. Used in conjunction with techniques such as proton magnetic resonance spectroscopy for the nonsurgical evaluation of SLNs, ICP-MS analysis of antimony concentrations in FNB samples could potentially serve as a minimally invasive alternative to surgery and histopathologic evaluation to objectively classify a given node as sentinel or nonsentinel and determine its tumor-harboring status.

Outcome in 846 cutaneous melanoma patients from a single center after a negative sentinel node biopsy.

BACKGROUND: A negative sentinel node biopsy (SNB) implies a good prognosis for melanoma patients. The purpose of this study was to determine the long-term outcome for melanoma patients with a negative SNB. METHODS: Survival and prognostic factors were analyzed for 836 SNB-negative patients. All patients with a node field recurrence were reviewed, and sentinel node (SN) tissue was reexamined. RESULTS: The median tumor thickness was 1.7 mm, and 23.8% were ulcerated. The median follow-up was 42.1 months. Melanoma specific survival at 5 years was 90%, compared with 56% for SN-positive patients (P < .001). On multivariate analysis, only thickness and ulceration retained significance for disease-free and disease-specific survival. Five-year survival for patients with nonulcerated lesions was 94% vs. 78% with ulceration. Eighty-three patients (9.9%) had a recurrence. Twenty-seven patients developed recurrence in the regional node field, and in 22 of these, it was the first recurrence site. Six developed local recurrence, 17 an in-transit metastasis, and 58 distant disease. The false-negative rate was 13.2%. SN slides and tissue blocks were further examined in 18 patients with recurrence in the node field, and metastatic disease was found in 3 of them. CONCLUSIONS: This large, single-center study confirms that patients with a negative SNB have a significantly better prognosis than those with positive SNs. In those with a negative SNB, primary tumor thickness and ulceration are independent predictors of survival. Incorrect pathologic diagnosis contributed to only a minority of the false-negative results in this study.

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.

Antimony concentrations in nodal tissue can confirm sentinel node identity.

The sentinel node biopsy procedure is a highly accurate method of staging patients with cutaneous melanoma and the tumor-harboring status of sentinel nodes is the most important prognostic factor. For the procedure to provide accurate prognostic information, however, it is essential that 'true' sentinel nodes are removed and examined thoroughly. A technique to confirm sentinel node identity may reduce the false-negative rate of the procedure. We have found that antimony (originating from the antimony sulfide colloid used for preoperative lymphoscintigraphy in our institution) can be measured in tissue sections of sentinel nodes using inductively coupled plasma mass spectrometry. The aims of this study were to determine whether antimony concentrations can be used to confirm that removed sentinel nodes are 'true' sentinel nodes and to differentiate sentinel nodes from nonsentinel nodes. In all, 24 patients who had both a tumor-positive sentinel node and a tumor-negative nonsentinel node removed from one regional node field during the same operation, were identified. Tissue sections (50 microm) thick were cut from archival paraffin blocks of each of the sentinel nodes and nonsentinel nodes. Antimony concentrations in the tissue sections were measured using inductively coupled plasma mass spectrometry. The median and mean concentrations of antimony in parts per billion were 0.526 and 1.198, respectively (range 0.020-7.596) in the sentinel nodes, and 0.043 and 0.123 (range 0-0.800) in the nonsentinel nodes (P = 0.004). In four of the 24 pairs, both the presumed sentinel nodes and the nonsentinel nodes had very low antimony levels (less than 0.18 parts per billion), suggesting that nodes designated as sentinel nodes may not have been 'true' sentinel nodes. It is concluded that determination of antimony concentrations within sentinel nodes using the highly sensitive method of inductively coupled plasma mass spectrometry can confirm the identity of sentinel nodes and validate the sentinel node technique.

Immunohistochemical stains fail to increase the detection rate of micrometastatic melanoma in completion regional lymph node dissection specimens.

In melanoma patients, examination of tissue sections stained for immunohistochemical markers as an adjunct to examination of haematoxylin and eosin (H&E)-stained sections has been shown to increase the detection rate of micrometastatic disease in sentinel lymph nodes (SLNs). However, immunohistochemical stains are not routinely performed on completion regional lymph node dissection (CLND) specimens in most centres and it is not known whether their use would increase the detection of micrometastatic disease in these specimens. This study was performed to determine whether the application of immunohistochemical stains for S100 and HMB45 (in addition to H&E stains) increases the detection of micrometastatic disease in CLND specimens of melanoma patients and whether their use would be cost-effective in routine pathological practice. Forty-nine CLND specimens from patients with a prior positive SLN biopsy were examined by performing H&E stains and immunohistochemical stains for S100 protein and HMB-45 on each node, and the detection rate of melanoma metastases using H&E-stained sections was compared with that using immunohistochemically stained sections. The number of nodes in the CLND specimens ranged from 4 to 37 (median 14, mean 14.7). Nodal deposits of melanoma cells were detected in 12 of 49 cases (24%). Among these 12 positive cases, the mean number of positive nodes per CLND specimen was 2.2 (range, 1-14). The total number of positive nodes in the 12 CLND specimens was 27, accounting for 3.8% of the 720 nodes removed in the study group of 49 cases. Melanoma cells in all 27 positive nodes were identified both on the H&E-stained slides and the slides stained immunohistochemically for S100. The melanoma cells were positive for HMB45 in 24 of the 27 lymph nodes that contained metastatic melanoma (the metastatic melanoma cells were negative for HMB45 in three positive nodes from one specimen). No further positive lymph nodes were detected with the immunostains that had not been identified on the H&E-stained sections. This study suggests that the use of immunostains does not increase the detection rate of metastatic melanoma in CLND specimens, and that their routine use would not be cost-effective. We therefore recommend only H&E staining on sections of all lymph nodes in CLND specimens from melanoma patients.

Parachordoma is not distinguishable from axial chordoma using immunohistochemistry.

Parachordoma is a rare soft tissue tumor that morphologically resembles chordoma of the axial skeleton but occurs in a peripheral site. A recent study reported immunohistochemical differences between chordoma and parachordoma. While both tumors were positive for cytokeratin (CK) 8/18 (as recognized by the antibody Cam5.2), S100 and epithelial membrane antigen (EMA), only the chordoma was positive for CK7, CK20, CK 1/5/10/14 (as recognized by the antibody 34betaE12) and carcinoembryonic antigen (CEA). It has since been suggested that tumors indistinguishable from chordoma that involve the periphery should be termed chordoma periphericum and that these tumors are distinct from parachordoma. In the current study, the clinical, radiological, pathological, immunohistochemical and ultrastructural features of a chordoma-like tumor involving the deep soft tissues of the lower leg of a 69-year-old woman are presented. Microscopically, the tumor had a pseudolobulated growth pattern and consisted of sheets, nests and cords of epithelioid cells, some with a physaliferous appearance, separated by abundant myxoid stroma. The tumor cells were positive for CK 8/18, EMA and S100, showed focal staining for CK7, and were negative for CK20, CK 1/5/10/14 and CEA. On the basis of these results a diagnosis of parachordoma was favored. For comparison, an immunohistochemical analysis of five axial chordomas was also performed. The chordomas showed positivity for CK 8/18 (5 of 5 cases), EMA (5 of 5 cases), S100 (5 of 5 cases), CK 1/5/10/14 (1 of 5 cases) and CK7 (1 of 5 cases). Stains for CK20 and CEA were negative in all five chordomas. The results of the present study suggest that the expression of antigens for CK 1/5/10/14, CK7, CK20 and CEA in chordoma might not be as common as what has been previously reported. The results also suggest that parachordoma might not be easily distinguished immunohistochemically from axial chordoma (and therefore also from so-called chordoma periphericum).

Failure to remove true sentinel nodes can cause failure of the sentinel node biopsy technique: evidence from antimony concentrations in false-negative sentinel nodes from melanoma patients.

We have recently found that antimony (originating from the technetium 99m antimony trisulfide colloid, used for preoperative lymphoscintigraphy) can be measured in tissue sections from archival paraffin blocks of sentinel nodes (SNs) by means of inductively coupled plasma mass spectrometry (ICP-MS) to confirm that removed nodes are true SNs. We performed a retrospective analysis of antimony concentrations in all our false-negative (FN) SNs to determine whether errors in lymphadenectomy (i.e., failure to remove true SNs) may be a cause of FN SN biopsies (SNBs). Among 27 patients with an FN SNB, metastases were found on histopathologic review of the original slides or additional sections in 7 of 23 patients for which they were available; however, antimony concentrations were low in 5 of 20 presumptive SNs. Our results suggest that an FN SNB can occur because of failure to remove the true SN as well as histopathologic misdiagnosis.

Interobserver reproducibility of histopathologic prognostic variables in primary cutaneous melanomas.

BACKGROUND: The prognosis for patients with localized primary cutaneous melanoma is known to depend principally on tumor thickness, and to a lesser extent on ulcerative state and Clark level. We have recently found in an analysis of 3661 patients that tumor mitotic rate (TMR) is also an important prognostic parameter, ranking second only to tumor thickness. However, few studies have assessed the accuracy and reproducibility with which these features of a melanoma are recorded by histopathologists. AIM: To assess interobserver reproducibility of major pathologic prognostic parameters in cutaneous melanoma. METHODS: Single hematoxylin and eosin-stained slides of 69 dermally invasive primary cutaneous melanomas were circulated among six pathologists with differing experience in the assessment of melanocytic tumors. The observers independently determined the tumor thickness, Clark level of invasion, ulcerative state, and TMR for each lesion. Intraclass correlation coefficients and kappa scores for multiple ratings per subject were calculated. RESULTS: The intraclass correlation coefficients were 0.96 for tumor thickness and 0.76 for TMR. The kappa scores were 0.83 for ulcerative state and 0.60 for Clark level. These results indicated excellent agreement among the pathologists for measurements of tumor thickness, ulcerative state, and TMR and fair to good agreement for Clark level. CONCLUSIONS: Appropriately trained and experienced histopathologists can assess prognostically important features of melanomas accurately and reproducibly. Given our recent finding of the significance of TMR in determining prognosis, it is important that this feature be assessed by a standardized method and documented for all primary cutaneous melanomas.

Use of multiple cytometric markers improves discrimination between benign and malignant melanocytic lesions: a study of DNA microdensitometry, karyometry, argyrophilic staining of nucleolar organizer regions and MIB1-Ki67 immunoreactivity.

Confident separation of benign naevi and malignant melanoma can sometimes be very difficult using conventional microscopy. This study evaluated the combined diagnostic abilities of multiple cytometric markers in separating various types of naevi from melanomas. The lesions studied included 27 benign compound naevi, 20 dysplastic naevi, 10 Spitz naevi and 24 melanomas. The cytometric features investigated were: (i) nuclear DNA content and chromatin compactness, measured by video imaged DNA microdensitometry; (ii) nuclear morphology, measured by nuclear morphometry (karyometry); (iii) transcriptional activity of nucleolar organizer regions, measured as the number and size of argyrophilic staining of nucleolar organizer regions (AgNORs); and (iv) cellular proliferative activity detected by quantifying the immunoreactivity of MIB1-Ki67 antigen. These variables were evaluated in the superficial, middle and deep zones of each lesion. Using multivariate discriminant analysis, a total diagnostic effectiveness of 97% could be achieved in separating the benign and malignant melanocytic lesions by co-evaluating variables for DNA microdensitometry, karyometry and AgNORs. A diagnostic effectiveness of 100% could be achieved if further co-evaluation with MIB1-Ki67 immunoreactivity was performed. Our study suggests that co-evaluation of multiple cytometric markers can improve the diagnostic abilities of individual techniques in separating benign naevi from malignant melanomas. This may be of particular significance in the diagnosis of melanocytic lesions whose biological behaviour cannot be confidently predicted by their histological features using conventional microscopy.

Pathologic review of negative sentinel lymph nodes in melanoma patients with regional recurrence: a clinicopathologic study of 1152 patients undergoing sentinel lymph node biopsy.

A sentinel lymph node (SLN) that is melanoma negative by pathologic examination implies absence of melanoma metastasis to that regional lymph node field. However, a small proportion of patients develop regional node field recurrence after a negative SLN biopsy. In this study, we reviewed the histopathology of negative SLNs from such patients to determine whether occult melanoma cells were present in the SLNs, to characterize the pathologic features of false-negative SLNs, and to provide recommendations for the histopathologic examination of these specimens. Between March 1992 and June 2001, of 1152 patients who had undergone SLN biopsy for primary melanomas at the Sydney Melanoma Unit, 976 were diagnosed with negative SLNs by initial pathologic examination (using 2 hematoxylin and eosin stained sections, and 2 immunostained sections for S-100 protein and HMB45), and follow-up was available in 957. Of these, 26 (2.7%) developed regional lymph node recurrence during a median follow-up period of 35.7 months. For 22 of them, the original slides and tissue blocks were available for reexamination. The original slides of each block were reviewed. Multiple further sections were cut from each block and stained with hematoxylin and eosin, for S-100, HMB45, and Melan A. Deposits of occult melanoma cells were detected in 7 of the 22 cases (31.8%). In 5 of the 7 cases, deposits of melanoma cells were present only in the recut sections. There were no significant differences in clinical and pathologic variables for those patients in whom occult melanoma cells were found by pathologic reexamination of their SLNs, compared with those in whom no melanoma cells were detected. The detection of melanoma cell deposits in only 7 of 22 false-negative SLNs suggests that mechanisms other than failure of histopathologic examination may contribute to the failure of the SLN biopsy technique in some patients. The failure rate for melanoma detection in SLNs by our routine pathologic examination, using the current protocol at our institution, was <1% (7 of 957 patients). Routinely performing more intensive histopathologic examination of SLNs is difficult to justify from a cost benefit perspective; we therefore recommend examining two hematoxylin and eosin stained sections and two immunostained sections (for S-100 and HMB45) routinely on SLNs from melanoma patients.

Argyrophilic staining of nucleolar organizer region count and morphometry in benign and malignant melanocytic lesions.

Differentiation between malignant melanomas (MMs) and benign nevi based on histologic features can sometimes be difficult. This study evaluated the diagnostic effectiveness of argyrophilic staining of nucleolar organizer regions (AgNORs) in separating benign nevi from MMs by assessing 27 compound nevi (CN), 20 dysplastic nevi (DN), 10 Spitz nevi (SN), and 24 MMs. Both AgNOR count and morphology variables were measured from the superficial, middle, and deep zones of the lesions using video image analysis. Malignant melanomas had a significantly greater AgNOR number per nucleus, mean AgNOR area per nucleus, and variation in AgNOR area per nucleus compared with all types of benign nevi (p < 0.05). In multivariate discriminant analysis using a combination of four AgNOR counts and morphometric parameters, all CN and DN, 8 of 10 SN, and 23 of 24 MMs could be correctly classified as benign or malignant. The results suggest that both AgNOR count and morphology help to separate benign and malignant melanocytic lesions and that the combination of both sets of parameters improves their discriminating ability.

Antimony by ICP-MS as a marker for sentinel lymph nodes in melanoma patients.

A sensitive, accurate and specific method for the analysis of antimony by ICP-MS is presented as a marker of the sentinel lymph node in melanoma patients.

Differentiating benign nevi from malignant melanoma using DNA microdensitometry and karyometry and maturation: a zonal comparison, correlation and multivariate analysis.

OBJECTIVE: To evaluate the diagnostic effectiveness of cytometric features of DNA microdensitometry, karyometry (nuclear morphometry) and maturation and their combinations in separating benign nevi from malignant melanomas. STUDY DESIGN: Tumor cells were measured from each of the superficial, middle and deep zones of 81 melanocytic lesions using video image analysis for nuclear DNA content, chromatin compactness, and nuclear size and shape variables. There were 27 banal compound melanocytic nevi, 20 dysplastic compound nevi, 10 Spitz nevi and 24 malignant melanomas (MM). Maturation of cells with depth into the dermis was also studied by comparing cells from superficial to deep zones. RESULTS: MM showed distinct characteristics of DNA microdensitometry, karyometry and maturation as compared to all groups of benign nevi. There were overall close correlations between nuclear DNA content variables and nuclear size parameters in the total group of 81 lesions. However, there were fewer significant correlations between the various indices in the group of melanomas alone. Using multivariate discriminant analysis, up to 97% of the lesions could be correctly separated as benign or malignant by a combination of five key microdensitometric, karyometric and maturation parameters. CONCLUSION: DNA microdensitometry, karyometry and maturation parameters have independent abilities in identifying individual malignant melanomas. Coevaluation of various cytometric features and maturation profiles offers better diagnostic ability in separating benign nevi from MM.

Melanin bleaching in argyrophilic staining of AgNORs in pigmented lesions: a morphometric evaluation.

OBJECTIVE: To establish a procedure that can effectively bleach melanin from pigmented lesions without affecting quantification of argyrophilic staining of nucleolar organizer regions (AgNORs). STUDY DESIGN: Twenty banal compound nevi, five from each of nonpigmented, slightly pigmented, moderately pigmented and heavily pigmented groups, were bleached by 10% H202 for periods of 0 (nonbleached controls) and 24 hours. AgNOR size and count parameters of nevomelanocytic nuclei were measured by video image analysis. Melanin bleaching using KMnO4 was also investigated. RESULTS: In all lesions treated with 10% H202 for 24 hours, the melanin was bleached effectively, with no qualitative change in AgNOR appearance. There were no significant differences in mean AgNOR number per nucleus (AgNOR number), mean individual AgNOR size (AgNOR size) or mean percentage of AgNOR area per nucleus (% nuclear area) between nonbleached and bleached sets in both the nonpigmented and slightly pigmented groups. However, disintegration of AgNOR dots was observed in those treated with 1% KMnO4 for 5, 10 and 15 minutes. There were significant decreases in AgNOR size (P = .002) and % nuclear area (P = .003) and increase in AgNOR number (P = .05) in the slightly pigmented group evaluated when treated with 1% KMnO4 for five minutes. CONCLUSION: Melanin in pigmented lesions can be bleached effectively with an H202 procedure without significantly affecting AgNOR staining properties in contrast to bleaching with KMnO4.