HMB-45 Antibody Research Articles

PRAME and Historical Immunohistochemical Antibodies Ki-67, P16, and HMB-45 in Ambiguous Melanocytic Tumors.

Ambiguous melanocytic lesions/tumors (AMLs) can be simply described as melanocytic neoplasms that cannot be differentiated as either a melanoma or a nevus. Preferentially expressed antigen in melanoma (PRAME) is a novel antibody that can help differentiate between nevi and melanomas. However, its usefulness remains controversial in AMLs. The aim of this study was to demonstrate the importance of PRAME and diagnostic auxiliary antibodies (Ki-67, p16, HMB-45) in the diagnosis of melanocytic lesions, especially in AMLs. This study included 52 ambiguous melanocytic lesions, 40 nevi, and 40 melanomas. All immunohistochemical studies were performed automatically using the Universal Alkaline Phosphatase Red Detection Kit. Different analytic approaches were used for each antibody based on the literature. Statistically, the multinomial forward stepwise elimination logistic regression analysis was used to create a statistical model to predict the diagnosis of melanocytic lesions based on clinical, morphological, and immunohistochemical data. PRAME positivity was very strong and diffuse in the melanoma group and statistically significantly higher than that of the AML and nevus groups. There was no statistically significant difference between the nevus and AML groups. The Ki-67 proliferation index and HMB-45 staining pattern provided valuable indications for distinguishing between these 3 groups. The P16 antibody was limited in supporting the differential diagnosis. Our statistical model showed that a high mitosis count, central pagetoid spread, and PRAME positivity increased the probability of melanoma against an AML diagnosis. This study showed the advantages of evaluating the PRAME antibody together with morphological features and other immunohistochemical markers (Ki-67 and HMB-45) in the differential diagnosis of melanocytic lesions.

A Fast and Automated Melanin-bleaching Method for Histopathologic Evaluation of Pigmented Melanoma Tissues.

Histopathologic examination of highly pigmented melanoma tissues has always been a challenge for pathologists. The high concentration of melanin pigment is an obstacle for immunohistochemistry and the ensuing evaluation. Therefore, removing melanin has become a crucial step for processing heavily pigmented melanoma samples. Several bleaching techniques have been proposed in the past, however, the most commonly used methods are time-consuming and poorly standardized. In this study, we propose a new fast and fully automated bleaching method applicable to validated immunohistochemical panels already used in the diagnosis of melanocytic tumors. The proposed bleaching protocol is based on sample pretreatment with 0.5% hydrogen peroxide and a Tris base pH 10 solution for 8 minutes at 80°C before antigen retrieval. Immunohistochemistry with HMB45, MART-1, Ki-67, SOX10, S-100, Tyrosinase, and BRAF(V600E) antibodies showed that this pretreatment removed excess melanin without affecting the tissue antigenicity and cytoarchitecture. In conclusion, we propose a new fast and automated bleaching protocol, easily transferable to a routine setting with efficient results in specimens in which the melanin pigmentation could blunt the histopathologic examination.

DYSPLASTIC MELANOCYTIC NEVUS IN THE ORAL CAVITY: MORPHOLOGIC AND IMMUNOHISTOCHEMICAL FINDINGS

A 35-year-old female patient attended the oral diagnostic service presenting with a lesion on the attached gingiva, in the region of dental elements 31 and 32. The lesion presented a red-blackish coloration, softened consistency, sessile implantation, and exophytic growth, measuring approximately 4 mm. An excisional biopsy was performed, and the diagnostic hypotheses of hemangioma and angiofibroma were considered. During microscopic examination, there was proliferation of nevus cells, organized in theques arrangements, exhibiting a certain degree of pleomorphism, hyperchromatism, and sometimes bizarre appearance. From the immunohistochemical analysis for HMB-45, Melan-A, S-100, and Ki-67 antibodies, the histopathologic diagnosis of dysplastic melanocytic nevus was given. This case emphasizes the importance of the association of clinical, histopathologic, and immunohistochemical characteristics to obtain an adequate diagnosis, to rule out any other lesions. A 35-year-old female patient attended the oral diagnostic service presenting with a lesion on the attached gingiva, in the region of dental elements 31 and 32. The lesion presented a red-blackish coloration, softened consistency, sessile implantation, and exophytic growth, measuring approximately 4 mm. An excisional biopsy was performed, and the diagnostic hypotheses of hemangioma and angiofibroma were considered. During microscopic examination, there was proliferation of nevus cells, organized in theques arrangements, exhibiting a certain degree of pleomorphism, hyperchromatism, and sometimes bizarre appearance. From the immunohistochemical analysis for HMB-45, Melan-A, S-100, and Ki-67 antibodies, the histopathologic diagnosis of dysplastic melanocytic nevus was given. This case emphasizes the importance of the association of clinical, histopathologic, and immunohistochemical characteristics to obtain an adequate diagnosis, to rule out any other lesions.

A cutaneous angiomyolipoma case mistaken for a parotid mass

Angiomyolipoma is a benign tumor consisting of vascular structures, smooth muscle and adipose tissue. It is most commonly found in the kidney and more infrequently in the liver, lung, heart, intestines, nasal cavity, oral cavity and ear. Cutaneous angiomyolipomas are very rare and show some differences with non-cutaneous angiomyolipomas, these are mostly located in the distal extremities and due to trauma, HMB-45 antibody negativity and appear more frequently in men. All cases except one presented until today were observed not to have cellular atypia; thus, cutaneous angiomyolipomas are considered not to perform malign transformation. It is thought that total excision of the mass is adequate in treatment of cutaneous angiomyolipoma. Our case applied to our clinic with a left preauricular mass which was thought to be a parotid mass after initial physical examination. After magnetic resonance imaging (MRI) scan was performed, it was seen that the mass did not originate from parotid gland After the FNAC resulted non-diagnostic, the mass was excised. Pathological examination revealed "angiomyolipoma" diagnosis.

Immunohistochemical Approach to the Differential Diagnosis of Meningiomas and Their Mimics.

The differential diagnosis between meningioma and others tumors can be challenging. This study aimed to evaluate different immunohistochemical markers for the differential diagnosis between meningioma and their morphological mimics. Immunohistochemistry was performed on tissue microarray with antiepithelial membrane antigen (EMA), progesterone receptor, somatostatin receptor 2A (SSTR2A), CD34, STAT6, S100, SOX10, HMB45, MelanA, GFAP, inhibin, and BCL2 antibodies. One hundred and twenty-seven meningiomas, 26 solitary fibrous tumor/hemangiopericytomas (SFT/HPC), 39 schwannomas, 17 hemangioblastomas, 21 melanomas, 9 gliosarcomas, 5 neurofibromas, 9 peripheral primitive neuroectodermal tumors, 7 synovial sarcomas, and 5 malignant peripheral nerve sheath tumors were included in the microarray. SSTR2A was the most sensitive (95.2%) and specific (92%) marker of meningiomas. In combination, SSTR2A and/or EMA positivity reached maximal sensitivity (100%). Coexpression of SSTR2A and EMA was the most specific (94.8%) for the diagnosis of meningioma, regardless of the grade or subtype, with the exception of the differential diagnosis with synovial sarcoma. All synovial sarcomas were EMA-positive and 6/7 SSTR2A-positive. STAT6 showed optimum sensitivity and specificity (100%) for SFT/HPC. SOX10 was the most sensitive (94.3%) and specific (100%) marker to discriminate meningiomas from schwannomas. In conclusion, SSTR2A, STAT6, and SOX10 were the most sensitive and specific markers to distinguish meningiomas from their morphological mimics.

Hepatic perivascular epithelioid cell neoplasm: A clinical and pathological experience in diagnosis and treatment.

Hepatic perivascular epithelioid cell neoplasm (PEComa) is a rare type of neoplasm derived from mesenchymal tumors that is often misdiagnosed as hepatocellular carcinoma (HCC), hepatic hemangioma or other liver malignancies. The clinical and histological characteristics of PEComa have yet to be fully documented. To optimize the diagnosis and treatment of the disease, a retrospective analysis was performed to investigate the clinicopathological characteristics of 7 patients diagnosed with hepatic PEComa in the Sun Yat-Sen Memorial Hospital between January 2004 and December 2015. Briefly, all the patients lacked specific symptoms, and a serological examination provided no further useful information. Additionally, non-specific imaging manifestations were observed. Microscopically, detection of epithelioid or spindle-shaped cells with adipocytes was suggestive of the disease, and an analysis of biomarkers, including the monoclonal antibody HMB-45, the melanocytic differentiation marker, Melan-A, and smooth muscle α-actin (SMA), helped to confirm the diagnosis. Regarding the treatment, 6 patients (85.7%) received surgical resection procedures, and 1 patient (14.3%) was admitted for tumor arterial embolization and percutaneous microwave coagulation therapy. After a follow-up period of 12–20 months, no recurrence was observed. Taken together, hepatic PEComa should be suspended in patients with a liver tumor with asymptomatic manifestation and normal serological test results. In imaging studies, hepatic PEComa was able to mimic HCC, hepatic hemangioma and metastatic tumor, although the mass did not invade the adjacent organs and vessel. The definitive diagnosis was made on the basis of the typical morphological features and notable markers of the tumor tissue. It is recommended that patients with PEComa in a benign pattern deserve serial imaging follow-up, but surgery is indicated in patients suffering from large tumors (>5 cm), or progressive enlargement or malignant tendency.

Histopathological characteristics of melasma

Objective To investigate histopathological and ultrastructural differences between melasma tissues and normal skin tissues around pigmented nevus. Methods Eight patients with melasma and 16 patients with facial pigmented nevus were included in this study. Two millimeter punch biopsies were taken from melasma lesions and adjacent normal skin of facial pigmented nevus. Biopsy specimens were then subjected to hematoxylin-eosin (HE) staining, Fonton-Masson staining, Verhoeff-van Gieson staining, and immunohistochemical staining with monoclonal antibodies HMB45 and NKI/beteb. Transmission electron microscopy was used to observe the tissue specimens. Semi-quantitative analysis was performed under a light microscope, and quantitative analysis by using a computerized image analysis system. Results Histopathological study revealed increased number of melanin granules mainly in the basal and prickle cell layers, sometimes in the dermis, in melasma tissues compared with normal skin tissues. Melanocytes were only observed in the epidermis of melasma tissues. Compared with normal skin tissues, melasma tissues showed no significant difference in the quantity of melanocytes, but a significant increase in the volume, staining intensity and dendrite number of melanocytes. In all of the 8 patients with melasma, mild to moderate lymphocytic infiltration was observed in the superficial dermis and around capillaries, with moderate telangiectasis in the superficial dermis. Electron microscopy revealed that there were more melanosomes in melanocytes and keratinocytes, and melanocyte dendrites extended into the dermis in melasma tissues. Conclusions Among the 8 patients, there were only two types of melasma, i.e., epidermal melasma and mixed melasma, and no dermal melasma was found. Inflammation and telangiectasis may induce or aggravate melasma. Key words: Chloasma; Pathology; Immunohistochemistry; Microscopy, electron, transmission; Inflammation; Telangiectasis

Increased Activity and Number of Epidermal Melanocytes in Lesional Psoriatic Skin

Background: Psoriatic lesions may resolve with hypo- or hyperpigmentation. The involvement of melanocytes in this dichotomous clinical outcome is not fully investigated. Objectives: Qualitative and quantitative assessment of melanocytes in untreated lesional and non-lesional psoriatic skin (n = 15) and healthy controls (n = 10). Methods: Skin biopsies were labelled immunohistochemically (APAAP technique) with the antimelanocyte monoclonal antibodies (MoAbs) HMB45, Melan A, tyrosinase and microphthalmia-associated transcription factor (MITF). The labelled melanocytes were evaluated by an independent investigator with a digital image analyser. Results: Lesional melanocytes, in contrast to those in non-lesional and healthy skin, exhibited features of activation in the form of dilatation, prominent and long dendrites and intense labelling. The number of melanocytes was significantly increased in psoriatic lesions in comparison with non-lesional psoriatic and healthy skin as shown by counts of cells labelled with the MoAbs HMB45 (3-fold; p < 0.001), Melan A (1.6-fold; p < 0.01) and tyrosinase (1.5-fold; p < 0.01). In contrast, labelling with MITF revealed no significant difference (1.2-fold increase; p > 0.05). Likewise, no significant difference between non-lesional psoriatic and healthy skin control was found (p > 0.05). Furthermore, no positively labelled dermal cells were detected, apart from few only detected with Melan A. Conclusions: Epidermal melanocyte activity and numbers are increased in the epidermal compartment of psoriatic lesions providing an explanation for postinflammatory hyperpigmentation.

Evidence Supporting a Lymphatic Endothelium Origin for Angiomyolipoma, a TSC2− Tumor Related to Lymphangioleiomyomatosis

Angiomyolipoma (AML) is a tumor closely related to lymphangioleiomyomatosis (LAM). Both entities are characterized by the proliferation of smooth muscle actin and melanocytic glycoprotein 100 (recognized by antibody HMB-45)-positive spindle-shaped and epithelioid cells. AML and LAM are etiologically linked to mutations in the tsc2 and tsc1 genes in the case of LAM. These genes encode the proteins tuberous sclerosis complex (TSC)-1 and TSC2, which are directly involved in suppressing the mechanistic target of rapamycin cell growth signaling pathway. Although significant progress has been made in characterizing and pharmacologically slowing the progression of AML and LAM with rapamycin, our understanding of their pathogenesis lacks an identified cell of origin. We used an AML-derived cell line to determine whether TSC2 restitution brings about the cell type from which AML arises. We found that AML cells express lymphatic endothelial cell markers consistent with lymphatic endothelial cell precursors invivo and invitro. Moreover, on TSC2 correction, AML cells mature into adult lymphatic endothelial cells and have functional attributes characteristic of this cell lineage, suggesting a lymphatic endothelial cell of origin for AML. These effects are dependent on TSC2-mediated mechanistic target of rapamycin inactivation. Finally, we demonstrate the invitro effectiveness of norcantharidin, a lymphangiogenesis inhibitor, as a potential co-adjuvant therapy in the treatment of AML.

Protein Expression Analysis of Melanocyte Differentiation Antigen TRP-2

Melanocyte differentiation antigens, such as gp100, tyrosinase, and Melan-A and their corresponding antibodies HMB45, T311, and A103, are major diagnostic tools in surgical pathology. Little is known about tyrosinase-related protein 2 (TRP-2, or dopachrome tautomerase/DCT) another melanocyte differentiation antigen, which is an enzymatic component of melanogenesis. We identified a commercial reagent to TRP-2, monoclonal antibody (mAb) C-9 and undertook a comprehensive analysis to assess its specificity and usefulness for surgical pathology. Subsequently, we analyzed panels of normal tissues and tumors. We show that TRP-2 is regularly expressed in melanocytes of the normal skin. In cutaneous nevi, TRP-2 is present in junctional as well as in dermal nevocytes. In malignant tumors, C-9 reactivity is restricted to melanocytic and related lesions and present in 84% and 58% of primary and metastatic melanomas, respectively. Ten primary melanomas of the anorectal mucosa were all positive. Like the other melanocyte differentiation antigens, TRP-2 was absent in 6 desmoplastic melanomas. Also, only 2 of 9 angiomyolipomas were TRP-2 positive. We conclude that mAb C-9 is a valuable reagent for the analysis of TRP-2 expression in archival surgical pathology material. The expression pattern of TRP-2 in melanocytic and related lesions appears to parallel other melanocyte differentiation antigens, although the overall incidence is lower than other antigens, such as Melan-A or gp100.

Antibody αPEP13h Reacts With Lymphangioleiomyomatosis Cells in Lung Nodules

Lymphangioleiomyomatosis (LAM) is characterized by the proliferation in the lung, axial lymphatics (eg, lymphangioleiomyomas), and kidney (eg, angiomyolipomas) of abnormal smooth muscle-like LAM cells, which express melanoma antigens such as Pmel17/gp100 and have dysfunctional tumor suppressor tuberous sclerosis complex (TSC) genes TSC2 or TSC1. Histopathologic diagnosis of LAM in lung specimens is based on identification of the Pmel17 protein with the monoclonal antibody HMB-45. We compared the sensitivity of HMB-45 to that of antipeptide antibody αPEP13h, which reacts with a C-terminal peptide of Pmel17. LAM lung nodules were laser-capture microdissected to identify proteins by Western blotting. HMB-45 recognized approximately 25% of LAM cells within the LAM lung nodules, whereas αPEP13h identified > 82% of LAM cells within these structures in approximately 90% of patients. Whereas HMB-45 reacted with epithelioid but not with spindle-shaped LAM cells, αPEP13h identified both spindle-shaped and epithelioid LAM cells, providing greater sensitivity for detection of all types of LAM cells. HMB-45 recognized Pmel17 in premelanosomal organelles; αPEP13h recognized proteins in the cytoplasm as well as in premelanosomal organelles. Both antibodies recognized a Pmel17 variant of approximately 50 kDa. Based on its sensitivity and specificity, αPEP13h may be useful in the diagnosis of LAM and more sensitive than HMB-45.

Extracellular matrix proteins, neural differentiation and melanoma progression

Background: Highly invasive melanoma cells traverse the extracellular matrix (ECM) by cell adhesion, ECM proteolysis, and cell migration. Ln5 and Integrin α5β1 play a key role in regulating melanoma growth, invasion, migration, angiogenesis and tissue remodeling. The present work utilizes the in situ 3D tumor matrix of melanomas to study their role in the formation of tumor-vascular-complexes (TVCs), vasculogenesis and tumor invasion. Material and Methods : Serial frozen and paraffin sections of nodular melanomas were subjected to histochemistry, enzyme histochemistry: dopa oxidase and immunohistochemistry using the monoclonal antibodies HMB45, GFAP, NFP, Syn, Ln5 (laminin 5) & integrin (α5β1) by the avidin/biotin system and assessed in general tumor areas, infiltrating margins, TVCs, vasculogenesis. Results : General areas: Ln5 shows a range of 75%-100% positivity in pigmented and 5.9%-29.8% cells in amelanotic areas. Integrin α5β1: shows a range of 69.4%-94.8% positivity in pigmented and 4%-55.2% in amelanotic areas. Infiltrating margins: Ln5/Integrin α5β1, are both required for preparing the ECM for spread of the tumor cells and angiogenesis. The cells at the infiltrating margins are positive for both. TVCs: Ln5/Integrin α5β1 expression is highest in the outer layers. Vasculogenesis (VM): Irregular spaces lined by Syn, Ln5/Integrin α5β1 positive tumor cells expand into islands of hemopoeisis, associated with the lining tumor cells. Combined TVCs and VM: In the mixed tumors a combination of TVCs and VM are seen. Irregular crevices appear lined by Ln5/Integrin α5β1 positive cells which flatten to form endothelial tubes which connect with the vascular network. Ln5/Integrin α5β1 positive cells co-express neural markers, NFP and Syn in all areas. Discussion: In conclusion, Ln5/Integrin α5β1 form the substrate for the advancing cell mass in the outer layers of the TVCs, infiltrating tumor margins and vasculogenic spaces. The architecture and remodeling of the TVCs is maintained with integrin α5β1 providing attachment and dynamic force for tissue compaction, cohesion and migration aided by Ln5. Neural differentiation is associated with migratory ability as indicated by NFP and Syn coexpression with Ln5/Integrin α5β1.

A Donor Cornea With Metastatic Cells From a Cutaneous Malignant Melanoma

To describe the case of a donor cornea that showed hematogenous metastatic spread of cutaneous melanoma to the sclerocorneal limbus. Corneal tissue obtained from a donor with cutaneous malignant melanoma was evaluated for endothelial cell density, corneal transparency, and epithelial morphology. Subsequently, hematoxylin and eosin staining and immunohistochemical characterization using S100, HMB45, Melan-A, and CD34 antibodies were performed on the corneal sections. The corneal tissue was transparent with high endothelial cell density; it was graded as being suitable for transplantation according to the current eye bank criteria. However, the aggressiveness of the donor's cancer and the diffuse melanosis of the sclera led to the suspicion of malignant melanoma metastasis to the cornea. Histochemical analysis of the corneoscleral rim showed small aggregates rich in pigmented cells that were localized in cleft-like structures in the sclera, at the sclerocorneal interface and in the peripheral avascular portion of the cornea. The aggregates were positive for the melanocytic tumor markers S100, HMB45, and Melan-A; the rims of the clefts expressed the panvascular CD34 antigen, which was suggestive of neovascularization. Corneal tissue from a donor with malignant cutaneous melanoma displayed neoplastic lesions of melanocytic origin that had spread from a primitive melanoma through hematogenous routes to the sclerocorneal limbus. On the basis of this finding, we believe that having a metastatic cutaneous malignant melanoma could in some cases be reviewed as an exclusionary criterion for undergoing cornea transplantation.

Marginal and Joint Distributions of S100, HMB-45, and Melan-A Across a Large Series of Cutaneous Melanomas

The distribution of the standard melanoma antibodies S100, HMB-45, and Melan-A has been extensively studied. Yet, the overlap in their expression is less well characterized. To determine the joint distributions of the classic melanoma markers and to determine if classification according to joint antigen expression has prognostic relevance. S100, HMB-45, and Melan-A were assayed by immunofluorescence-based immunohistochemistry on a large tissue microarray of 212 cutaneous melanoma primary tumors and 341 metastases. Positive expression for each antigen required display of immunoreactivity for at least 25% of melanoma cells. Marginal and joint distributions were determined across all markers. Bivariate associations with established clinicopathologic covariates and melanoma-specific survival analyses were conducted. Of 322 assayable melanomas, 295 (91.6%), 203 (63.0%), and 236 (73.3%) stained with S100, HMB-45, and Melan-A, respectively. Twenty-seven melanomas, representing a diverse set of histopathologic profiles, were S100 negative. Coexpression of all 3 antibodies was observed in 160 melanomas (49.7%). Intensity of endogenous melanin pigment did not confound immunolabeling. Among primary tumors, associations with clinicopathologic parameters revealed a significant relationship only between HMB-45 and microsatellitosis (P = .02). No significant differences among clinicopathologic criteria were observed across the HMB-45/Melan-A joint distribution categories. Neither marginal HMB-45 (P = .56) nor Melan-A (P = .81), or their joint distributions (P = .88), was associated with melanoma-specific survival. Comprehensive characterization of the marginal and joint distributions for S100, HMB-45, and Melan-A across a large series of cutaneous melanomas revealed diversity of expression across this group of antigens. However, these immunohistochemically defined subclasses of melanomas do not significantly differ according to clinicopathologic correlates or outcome.

LAM cells biology and lymphangioleiomyomatosis

Progressive lung tissue destruction in lymphangioleiomyomatosis (LAM) occurs as a result of excessive proliferation of LAM cells caused by a mutation in one of the tuberous sclerosis complex suppressor genes, TSC1 or TSC2. These cells show constitutive activation of the mammalian target of rapamycin (mTOR) pathway and many of the mTOR-related kinases such as Akt, Erk, S6K1 and S6. Phenotype of LAM cells differs considerably depending on their microenvironment. LAM cells show differences in morphology, size and expression of various factors depending on their location in the tumor or body fluids. The presence of LAM cells in blood, urine, bronchoalveolar lavage fluid (BALF), and chyle proves their ability to metastasis. Antigens of smooth muscle cells are expressed in most LAM cells. Some of these cells are immunoreactive with HMB-45 antibody, which is used for the immunohistochemical diagnosis of LAM. Receptors for estrogen and progesterone may also be expressed in these cells, which probably is associated with the fact that LAM occurs almost exclusively in women of childbearing age. LAM cells via increased production of metalloproteinases are involved in the destruction of the extracellular matrix, as well as the remodeling and damage of lung tissue. Sporadic LAM occurs extremely rarely. Therefore a good experimental model of this disease is necessary. To date, several animal and human cell lines, which both genetically and phenotypically resemble LAM cells, have been obtained. These cell lines, derived from LAM nodule or an angiomyolipoma, are usually characterized by a mutation of the TSC2 gene, expression of smooth muscle cell antigens such as a-smooth muscle actin (aSMA) or S6K1 and S6 protein hyperphosphorylation. Presently, there is no commercially available cell line representing a good model of LAM. A better understanding of LAM cell biology is necessary for creating a useful model in vitro for further exploration of both LAM pathomechanisms and more general mechanisms of carcinogenesis.

Primary amelanotic melanoma of the rectum mimicking adenocarcinoma

Patient: Male, 55Final Diagnosis: MelanomaSymptoms: Worsening constipation • tenesmus • weight lossMedication: —Clinical Procedure: Chemoradiation therapySpecialty: OncologyObjective:Challenging differential diagnosisBackground:Malignant melanoma is usually readily diagnosed by the presence of melanin granules. Although amelanotic melanoma contains a few melanin granules, it is often difficult to differentiate from non-epithelial malignant tumors. Immunohistochemical staining may be needed to diagnose the condition.Case Report:This report describes a case of amelanotic melanoma of the rectum, which was originally suspected to be an adenocarcinoma, but was subsequently correctly diagnosed by immunohistochemical staining with HMB-45 antibody and by the presence of S-100 protein. A pinkish-red ulceroproliferative growth was located about 7 cm from the anal verge. The patient was treated by laparoscopic low anterior resection.Conclusions:Very few cases of amelanotic melanoma of rectum have been reported in the literature and there is only limited clinical experience with this disease. It appears to be a highly lethal tumor and may need much more aggressive treatment than that used for carcinoma of the rectum.

Disseminated tumour cells in bone marrow of patients with uveal melanoma

Approximately 50% of patients with uveal melanomas develop metastases. Thus, it is important to improve our understanding of how melanoma metastases develop. As part of a uveal melanoma micrometastasis study, we compared the detection rates of immunomagnetically selected (IMS) tumour cells in bone marrow (BM) with positively stained tumour cells using immunocytochemistry (ICC). Bone marrow mononuclear cells were isolated. Immunocytochemistry cytospin preparations were immunocytochemically stained in parallel with two different melanoma antibodies, 9.2.27 and HMB45. Using IMS, melanoma cells were selected from BM mononuclear cell fractions using immunomagnetic beads coated with the 9.2.27 antibody and identified by light microscopy. In cytospin preparations from 226 patients, melanoma cells were detected in 24 (10.6%), 10 with 9.2.27 and 17 with the HMB45 antibody. In three cases, we found positive cells with both antibodies. Six of the 226 (2.6%) patients that stained positively with ICC died with metastatic disease, all also positive with IMS. Sixty-six (29.2%) patients had positive BM samples with IMS at the first examination. Immunomagnetic selection (IMS) was positive in 36.8% of the 57 patients who later developed clinical metastases. Twenty-one IMS-positive patients and 31 IMS-negative patients died of metastases, in total 52 of 226 patients (23.0%). The mortality rate of melanoma metastasis was 24% (6/24) after at least 4 ½ years in ICC-positive patients compared to 38.5% (20/52) in IMS-positive patients. The presence of melanoma cells in BM of patients with uveal melanoma is documented in our study with IMS and ICC. Immunomagnetically selected is more sensitive than ICC in detecting tumour cells in BM. However, statistically, we did not find any prognostic impact of the presence of melanoma cells in BM.

Analysis of BRAF mutations in circulating tumor cells selected by size from patients with melanoma and comparision to the primary tumor.

e21014 Background: The efficacy of anti-BRAF therapies in patients with melanoma has emphasized the need to identify BRAF mutations in the tumour. As circulating tumour cells (CTC) derive from the primary tumour (PT), their analysis may represent a non-invasive tool to follow the molecular-target in blood after tumour removal. Methods: This prospective study included 26 patients: 24 patients had confirmed melanoma, 2 patients had benign lesions (1 naevus and 1 pyogenic granuloma). Blood samples were taken at time of the PT surgical excision; 12 healthy donors were included as controls. The 24 patients, 13 females and 11 males, had invasive melanoma (13 superficial spreading melanoma, 7 nodular melanoma, 1 lentigo malignant melanoma, 1 desmoplastic melanoma, 2 unclassified melanoma.). The mean thickness was 7.8 mm. Cytology, immunophenotyping and analysis of BRAF mutations on exon 15 were performed comparatively in primary tumour and CTC from the same patient. CTC were selected by the ScreenCell method, a single-use innovative system to isolate rare tumour cells through a filter-membrane on the basis of their size. Nucleic acids DNA/RNA were extracted from live CTC after filtration. Results: Isolated CTC and microemboli were found in 21/24 patients and were found comparable to the primary melanoma cells. No CTC was found in 10 healthy donors and 2 patients with benign lesions. The mean number of CTC was 2 CTC per ml, the maximum was 4 CTC and 7 microemboli per ml. Malignancy of CTC was assessed on classic criteria: cell size larger than 15 microns, nucleo-cytoplasmic ratio &gt; 0.5, presence of clusters composed of pleomorphic cells. CTC were positively immunostained either by S-100 or MART1/melanA or HMB45 antibodies; 4 patients were studied for BRAF mutations in both melanoma and CTC. The results (2 negative and 2 positive) were concordant in primary melanoma cells and CTC. The V600E-BRAF mutation was found in the 2 positive cases. Conclusions: The ScreenCell method of CTC selection by size allows to detect BRAF mutations in CTC of patients with melanoma. This simple method could be proposed to follow the molecular-target during the course of evolution and treatment after tumour removal.

MR Imaging Findings of the Intraspinal Meningeal Melanocytoma: Correlation with Histopathologic Findings

Our aim was to better understand and improve the accuracy of the preoperative diagnosis of intraspinal MM by a combined analysis of MR imaging and pathologic findings. All 5 patients had undergone unenhanced and contrast-enhanced MR imaging examinations. All tumor samples had immunohistochemical reactions to HMB-45, vimentin, S-100, EMA, and Leu-7 antibodies. All 5 cases were located in the intradural extramedullary compartment. Two cases had multifocal lesions, and 3 cases were solitary. Two cases showed homogeneously strong enhancement, and 3 cases showed moderate enhancement on contrast-enhanced T1WI. The tumor cells had positive reactions to HMB-45, vimentin, and S-100 antibodies. MR imaging plays an important role in the detection and diagnosis of intraspinal MM. Final diagnosis should be based on histopathology and IHC examinations.

Aberrant expression of HMB-45 in traumatized melanocytic nevi

Assessment of histologic and immunohistochemical maturation (with HMB-45 and anti-Ki-67) may be helpful in differentiating benign melanocytic nevi (BMN) from malignant melanoma. Recently, we reported loss of maturation and aberrant immunohistochemical findings in melanocytic nevi after liquid nitrogen cryotherapy (Adeniran etal, J Am Acad Dermatol 2009;61:341-5). Herein we report a similar phenomenon identified in traumatized melanocytic nevi (TMN). We sought to evaluate the histologic and immunohistochemical findings in early and late stages of traumatized nevi. Twenty-four cases of TMN were retrieved from the pathology archives. These were then assessed by two pathologists (T.L. and A.H.D.) using HMB-45 and MIB-1 (for Ki-67) antibodies. TMN showed some of the following findings: epidermal changes (parakeratosis, ulceration, serum crust, flattening of the epidermis) and dermal changes including fibrosis and the presence of melanophages. In some cases, there was architectural disorder of the overlying melanocytes, with crowding in the basal layer, but without significant pagetoid spread. Occasionally, the dermal scar contained larger, more epithelioid-appearing melanocytes than those beneath the scar. Fifty-four percent of TMN lacked obvious immunohistochemical maturation with HMB-45, since nevus cells within the scar or directly beneath it were strongly labeled. None of the TMN showed appreciable labeling for Ki-67. The exact clinical duration between trauma and biopsy could not be determined. Loss of maturation with HMB-45 in TMN can be a diagnostic pitfall in challenging cases. Concurrent evaluation of MIB-1 expression, along with the characteristic histologic features of trauma, should allow the correct diagnosis to be reached.