Unequivocal Melanomas Research Articles

Diffuse PRAME Expression Is Highly Specific for Thin Melanomas in the Distinction from Severely Dysplastic Nevi but Does Not Distinguish Metastasizing from Non-Metastasizing Thin Melanomas.

Simple SummaryHistological diagnoses within the spectrum from moderately dysplastic nevi to thin melanomas are neither accurate nor reproducible, emphasizing the need for more objective supplemental immunohistochemical markers. PRAME immunohistochemistry aids in differentiating unequivocal melanomas from unequivocal nevi. The aim of our study was to determine whether PRAME IHC also allows differentiation of severely dysplastic nevi from thin melanomas and whether PRAME is of prognostic significance in thin melanomas. We studied 70 thin melanomas, of which 35 metastasized and 35 did not metastasize and 35 severely dysplastic nevi. We found that diffuse PRAME expression was highly specific but only moderately sensitive for thin melanomas. Melanomas and severely dysplastic nevi with PRAME immunoreactivity had different staining patterns. Most Melanomas demonstrated diffuse PRAME staining of intraepidermal and dermal melanocytes while most severely dysplastic nevi showed a decreasing gradient with depth. PRAME did not allow for the differentiation of metastasizing and non-metastasizing melanomas.Background: PReferentially expressed Antigen in MElanoma (PRAME) immunohistochemistry is increasingly used as diagnostic adjunct in the evaluation of melanocytic tumors. The expression and prognostic significance of PRAME in melanomas ≤1.0 mm and its diagnostic utility in the distinction from severely dysplastic compound nevi (SDN) have not been studied. Methods: We investigated and compared the immunohistochemical PRAME expression in 70 matched thin metastasizing and non-metastasizing melanomas and 45 nevi from patients with long-term follow-up (35 SDN and 10 unequivocally benign compound nevi). Results: Diffuse PRAME staining in >75% of lesional epidermal and dermal melanocytes identified 58.6% of thin melanomas but did not distinguish metastasizing from non-metastasizing melanomas (p = 0.81). A superficial atypical melanocytic proliferation of uncertain significance, in which the final diagnostic interpretation favored a SDN was the only nevus with diffuse PRAME expression (1/45). Melanomas and SDN with PRAME immunoreactivity exhibited different staining patterns. Most melanomas (67.6%) showed uniform PRAME expression in the in situ and invasive component, whereas most SDN (81.0%) showed a decreasing gradient with depth. Conclusion: Diffuse intraepidermal and dermal PRAME staining is highly specific for melanomas in the distinction from SDN. PRAME expression is not a prognostic biomarker in melanomas ≤1.0 mm.

The utility of PRAME immunohistochemistry in the evaluation of challenging melanocytic tumors.

PRAME (PReferentially expressed Antigen in Melanoma) immunohistochemistry has demonstrated high specificity for unequivocal melanomas; however, its utility in ambiguous melanocytic neoplasms has yet to be fully elucidated. Cases of challenging melanocytic neoplasms were subclassified into one of three categories: challenging, favor benign (FB), challenging, cannot be subclassified (CCS), or challenging, favor malignant (FM). Using a previously published system, whereby cases with diffuse staining (>75%) were considered positive, scoring of PRAME was performed. Additionally, tumors with hotspot staining were also considered positive. Sixteen out of 85 tumors showed positive staining representing 5% of FB tumors, 24% of CCS tumors, and 47% of FM. In FB and CCS tumors, positive staining was mainly encountered in atypical intraepidermal melanocytic proliferations and spitzoid neoplasms. The specificity of positive PRAME staining was 95% and its concordance with the final diagnostic interpretation was 75%. PRAME positivity is more common in neoplasms favored to be malignant by histopathologic evaluation. Its clinical utility may include early diagnosis of incipient melanoma in situ. Rarely, benign melanocytic neoplasms could show diffuse expression of PRAME, and additional studies are needed to determine optimal utilization. Lastly, hotspot staining may increase its sensitivity without much compromise in specificity.

Mohs micrographic surgery with melanoma antigen recognized by T cells 1 (MART-1) immunostaining for atypical intraepidermal melanocytic proliferation

The efficacy of Mohs micrographic surgery (MMS) for atypical intraepidermal melanocytic proliferation (AIMP) is unknown. To ascertain the frequency of diagnostic change to melanoma (upstaging) and the frequency of local recurrence after MMS for AIMP. A secondary outcome was the frequency of subclinical spread (defined as the requirement for >1 stage of MMS to achieve tumor-free margins). Retrospective, cross-sectional study of 223 AIMP (with 92.4% located on the head, neck, hand, foot, or pretibial leg) patients treated with MMS with melanoma antigen recognized by T cells 1 (MART-1) immunostaining. Upstaging to unequivocal melanoma in situ or invasive melanoma was identified in 18.8% (42/223) of all AIMP patients. The local recurrence rate was 0% (0/223) with a mean follow-up time of 2.7years (998days). Subclinical spread was present in 23.8% (53/223) of AIMP patients. Single site, retrospective design, observational study, lack of objective criteria to diagnose AIMP. MMS with MART-1 immunostaining achieves excellent local control of specialty site AIMP and permits definitive removal of subclinical spread before reconstruction. The central debulking excision should be evaluated with formalin-fixed paraffin-embedded section staining, since a significant percentage of AIMP are reclassified as melanoma in situ or invasive melanoma.

A diagnostic algorithm for atypical spitzoid tumors: guidelines for immunohistochemical and molecular assessment

Atypical spitzoid tumors are a morphologically diverse group of rare melanocytic lesions most frequently seen in children and young adults. As atypical spitzoid tumors bear striking resemblance to Spitz nevus and spitzoid melanomas clinically and histopathologically, it is crucial to determine its malignant potential and predict its clinical behavior. To date, many researchers have attempted to differentiate atypical spitzoid tumors from unequivocal melanomas based on morphological, immonohistochemical, and molecular diagnostic differences. A diagnostic algorithm is proposed here to assess the malignant potential of atypical spitzoid tumors by using a combination of immunohistochemical and cytogenetic/molecular tests. Together with classical morphological evaluation, this algorithm includes a set of immunohistochemistry assays (p16Ink4a, a dual-color Ki67/MART-1, and HMB45), fluorescence in situ hybridization (FISH) with five probes (6p25, 8q24, 11q13, CEN9, and 9p21), and an array-based comparative genomic hybridization. This review discusses details of the algorithm, the rationale of each test used in the algorithm, and utility of this algorithm in routine dermatopathology practice. This algorithmic approach will provide a comprehensive diagnostic tool that complements conventional histological criteria and will significantly contribute to improve the diagnosis and prediction of the clinical behavior of atypical spitzoid tumors.

Deep penetrating nevus-like borderline tumors: A unique subset of ambiguous melanocytic tumors with malignant potential and normal cytogenetics.

Deep penetrating nevi (DPN) are a relatively uncommon subtype of melanocytic nevi. A small subset of these lesions exhibit atypical features (cytologic and architectural atypia, mitotic activity) seen in melanoma. These lesions we term the deep penetrating nevus-like borderline tumor. Unequivocal melanomas can show overlapping morphologic features of DPN, which have been termed plexiform melanomas. 40 cases of DPN-like borderline tumor were identified along with 6 cases of plexiform melanoma. Clinical follow up was obtained, along with cytogenetic analysis in the form of fluorescent in situ hybridization (FISH) and/or comparative genomic hybridization (CGH). The DPN-like borderline tumor cases included 24 females and 16 males. Of sentinel lymph node biopsies performed, 1/3 of cases showed lymph node involvement. All patients where an aggressive clinical approach was adopted remain free of disease. All 6 DPN-like borderline tumor cases tested by CGH showed normal cytogenetics, as did 7 of 9 cases tested by FISH. Of the plexiform melanomas, 4/6 patients died of disease. In 3 cases there was morphologic progression from a DPN-like borderline tumor to overt melanoma. In one case of progression, cytogenetics was normal in the DPN-like borderline tumor and then abnormal in the progressed melanoma. DPN-like borderline tumors are melanocytic tumors associated with a high incidence of regional lymph node disease and exhibiting the potential for melanoma progression despite a normal cytogenetic profile. Patients with these lesions should be aggressively managed, with at least complete re-excision and consideration of sentinel node biopsy, regardless of cytogenetic data.

Fluorescence In Situ Hybridization as an Ancillary Tool in the Diagnosis of Ambiguous Melanocytic Neoplasms

Previous studies have demonstrated the utility of fluorescence in situ hybridization (FISH) as an ancillary method in the diagnostic workup of histopathologically ambiguous melanocytic neoplasms. A combination of probes targeting 3 loci on chromosome 6 and 1 on 11q has been reported to distinguish unequivocal melanomas and nevi with a sensitivity and specificity of 87% and 96%, respectively. However, information on how FISH should be integrated into routine clinical testing is limited. We report our experience of FISH testing of 804 ambiguous melanocytic lesions performed as part of routine workup at University of California, San Francisco. The main category (47% of all cases) for which FISH testing was requested was Spitz tumors. Other categories included the distinction of possible melanoma from combined nevi (9%), acral or mucosal nevi (9%), Clark/dysplastic nevi (7%), and blue or deep penetrating nevi (6%) and to assess the possibility of nevoid melanoma (4%). Of the ambiguous tumors successfully tested, 88% received a more definitive benign or malignant final diagnosis. Of the 630 cases that tested negative by FISH, the final diagnosis was benign in 489 (78%) cases, ambiguous in 91 cases (14%), and malignant in 50 cases (8%). A positive FISH result was observed in 124 cases, with a final diagnosis of melanoma in 117 (94%). One (1%) FISH-positive case had an equivocal final diagnosis, and 6 (5%) were interpreted, despite the positive FISH result, as melanocytic nevi. We conclude that FISH testing can help reduce the number of equivocal diagnoses in ambiguous melanocytic neoplasms, in particular if FISH testing is positive, and discuss the challenges and limitations of FISH in clinical practice.

What's new in prognostication of melanoma in the dermatopathology laboratory?

With the advent of genetic and epigenetic research, molecular techniques could someday be used to discriminate nevus from melanoma so that ambiguous melanocytic lesions could be more accurately classified or that prognostication could be improved in melanoma patients. That promised day might be closer than realized. The last 20 years of research in cytogenetic and genetic alterations in melanoma have culminated in defined chromosomal lesions discriminating benign from malignant melanocytic tumors. Exploiting these differences, fluorescence in situ hybridization (FISH) can reproducibly discriminate unequivocal melanomas from melanocytic nevi with high sensitivity and specificity. The discriminating power of FISH in melanocytic tumors with ambiguous histopathology is questionable, however, because there is no standard definition of “malignancy.” Additional FISH studies on ambiguous cases are needed through international collaborations where large collections of such cases are shared and the “proof of malignancy” is established by adequate clinical follow-up. This contribution reviews the diagnostic utility of DNA-based FISH technology as it compares the diagnostic accuracy in melanocytic tumors with unambiguous vs ambiguous histopathology. The melanoma epigenome is further characterized through research into various activities of small interfering RNAs, such as microRNAs, providing the pathway for the application of microRNA-based strategies that could be the basis for future diagnostic biomarkers and molecular therapies in melanoma.

A Proposal for Improving Multicolor FISH Sensitivity in the Diagnosis of Malignant Melanoma Using New Combined Criteria

Fluorescence in situ hybridization (FISH) for the diagnosis of melanoma makes use of specific fluorescent probes to detect selected chromosomal alterations on paraffin-embedded tissue samples. To date, interpretation of FISH data has been based on numerical values generated by 2 different computational algorithms that of Abbott and that of Gerami. To further evaluate the value of FISH in the diagnosis of malignant melanoma, we selected 163 clinically and histologically unequivocal cases of malignant melanoma in a cohort of 575 melanocytic tumors and analyzed FISH data using the criteria of Abbott, Gerami, and new combined criteria. Depending on the used criteria, FISH was positive in the unequivocal malignant melanoma in 69.3% (113/163) of cases using the Abbott criteria, 74.2% (121/163) of cases using the Gerami criteria, and 82.2% (134/163) of cases using the combined criteria of Abbott and Gerami. Although use of all 3 criteria was associated with 100% FISH negativity in a cohort of 30 unequivocal benign melanocytic nevi, use of the combined criteria revealed more FISH-positive cases in ambiguous benign melanocytic lesions than the criteria of Abbott or Gerami alone: Abbott, 125 of 367; Gerami, 146 of 367; combined, 161 of 367. Furthermore, we show that 66% (8/12) of FISH-negative cases of unequivocal melanoma are positive when analyzed by array comparative genomic hybridization (aCGH), demonstrating that false-negative results remain despite the usage of the combined criteria for evaluation of FISH data. In these 8 FISH-negative aCGH-positive cases, copy number alterations were often located on chromosomes 9p, a chromosomal locus that is not targeted by the FISH probes currently used. In conclusion, the existing criteria for the evaluation of multicolor melanocytic FISH are limited by a nonnegligeable rate of false negativity that can be reduced by using newly proposed combined criteria but at the cost of increased detection of FISH positivity in ambiguous benign melanocytic lesions.

A Highly Specific and Discriminatory FISH Assay for Distinguishing Between Benign and Malignant Melanocytic Neoplasms

The diagnosis of certain melanocytic proliferations remains one of the most challenging areas in pathology. In recent times, fluorescence in situ hybridization (FISH) has emerged as a promising diagnostic aid to conventional microscopy. We previously showed that a 4-probe FISH assay targeting 6p25 (RREB1), 6q23 (MYB), Cep6 (centromere 6), and 11q13 (CCND1) could discriminate between histologically unequivocal melanomas and benign nevi with a sensitivity of 86.7% and specificity of 95.4%. However, the sensitivity of the assay is approximately 70% in melanomas with spitzoid morphology. Furthermore, differentiating true gains from tetraploidy may cause difficulties in interpretation by inexperienced examiners. Here we refine the current probe set to better target spitzoid melanomas and more easily distinguish cells with imbalanced copy number aberrations from tetraploid cells. Using FISH data from 3 training sets of 322 tumors, including 152 melanomas and 170 nevi, we identified 9p21, 6p25, 11q13, and 8q24 as a probe set with improved discriminatory power in differentiating melanomas from nevi. In a validation set of 51 melanomas and 51 nevi this probe set had a sensitivity of 94% and specificity of 98%, compared with the original probe set that had a sensitivity of 75% and specificity of 96% in the same validation cohort. We propose that by incorporating 9p21 into the 4-probe FISH assay, with a new diagnostic algorithm, this new probe set would have improved discriminatory power in melanocytic neoplasms and improved sensitivity for detecting spitzoid melanomas, as demonstrated by our previous studies.

Melanoma-Specific Marker Expression in Skin Biopsy Tissues as a Tool to Facilitate Melanoma Diagnosis

Diagnosis of cutaneous melanoma requires accurate differentiation of true malignant tumors from highly atypical lesions, which lack the capacity to develop uncontrolled proliferation and to metastasize. We used melanoma markers from previous work to differentiate benign and atypical lesions from melanoma using paraffin-embedded tissue. This critical step in diagnosis generates the most uncertainty and discrepancy between dermatopathologists. A total of 193 biopsy tissues were selected: 47 melanomas, 48 benign nevi, and 98 atypical/suspicious, including 48 atypical nevi and 50 melanomas as later assigned by expert dermatopathologists. Performance for SILV, GDF15, and L1CAM normalized to TYR in unequivocal melanoma versus benign nevi resulted in an area under the curve (AUC) of 0.94, 0.67, and 0.5, respectively. SILV also differentiated atypical cases classified as melanoma from atypical nevi with an AUC=0.74. Furthermore, SILV showed a significant difference between suspicious melanoma and each suspicious atypia group: melanoma versus severe atypia and melanoma versus moderate atypia had P-values of 0.0077 and 0.0009, respectively. SILV showed clear discrimination between melanoma and benign unequivocal cases as well as between different atypia subgroups in the group of suspicious samples. The role and potential utility of this molecular assay as an adjunct to the morphological diagnosis of melanoma are discussed.

Use of Fluorescence In situ Hybridization (FISH) to Distinguish Intranodal Nevus From Metastatic Melanoma

With the increase in sentinel lymph node biopsies in melanoma patients, pathologists are frequently confronted with small deposits of morphologically bland melanocytes in the node, which occasionally cannot be readily classified as benign nodal nevi or melanoma. As most melanomas harbor characteristic chromosomal aberrations which can be used to distinguish them from benign nevi, we used fluorescence in-situ hybridization (FISH) with markers for 3 regions on chromosome 6 and 1 on chromosome 11 to determine the presence of chromosomal aberrations in sentinel lymph node specimens with small foci of melanocytes that had been diagnosed as metastatic melanoma or nodal nevi by histopathology. Fifty-nine tissue samples from 41 patients (24 lymph node metastases, 17 with nodal nevi, and 18 of the available corresponding primary melanomas) were analyzed by FISH. Twenty of 24 (83%) cases diagnosed as metastatic melanoma showed aberrations by FISH. Of the 4 negative cases, 3 were unequivocal melanoma metastases, whereas 1 on re-review was histopathologically equivocal. Of the 17 nodal nevi, 1 (6%) also showed aberrations by FISH, whereas the remainder was negative. Multiple aberrations were present in the positive case, some of which were also found in the corresponding primary tumor, suggesting that this case represents a deceptively bland melanoma metastasis that had been misclassified by histomorphology. Our data indicate that FISH is a useful adjunct tool to traditional methods in the diagnostic workup of deposits of melanocytes in lymph nodes that are histopathologically ambiguous.

Consumption of the Epidermis

The distinction between melanoma and its most important simulant, Spitz nevus, is usually made on microscopically. We point out "consumption of the epidermis" (COE) as an additional diagnostic criterion. We defined COE as thinning of the epidermis with attenuation of the basal and suprabasal layers and loss of rete ridges in areas of direct contact with neoplastic melanocytes. We analyzed 102 unequivocal melanomas and 125 unequivocal Spitz nevi for the presence of COE. COE had not been used in arriving at the diagnosis of these cases because we were unaware of the criterion at the time that the cases were first evaluated. COE was found in 88 of 102 (86%) of melanomas but only 12 of 125 (9.6%) of Spitz nevi (P < 0.001). We then looked for COE in an independent set of 61 ambiguous melanocytic lesions with overlapping histopathologic features that could not be classified unequivocally as Spitz nevus or melanoma. The cases were analyzed by comparative genomic hybridization (CGH) for aberration patterns suggesting a benign or a malignant process, based on previous studies. COE was found in only 6 of 42 (14%) of the ambiguous cases in which CGH suggested a benign process and 14 of 19 (74%) of the ambiguous cases in which CGH suggested melanoma (P < 0.001). Our data suggest that COE is a useful criterion in the evaluation of melanocytic neoplasms. Because COE was frequently found at the edges of ulcers in the majority of ulcerated melanomas, the thinning of the epidermis in COE may represent an early phase of ulceration. This may prove to be important in distinguishing ulceration due to an effect of the tumor from ulceration due to trauma, which would be expected not to have the same prognostic import. Future studies are required to analyze the prognostic value of COE itself.

High-risk surgically resected pediatric melanoma and adjuvant interferon therapy.

Pediatric patients with high-risk surgically resected melanoma are at risk for relapse, yet little is known about these young patients and how they tolerate high-dose interferon therapy. We reviewed medical records of patients (< or =18 years) with high-risk melanoma referred to the University of Michigan Pediatric Hematology-Oncology service between January 1989 and July 2003. Fourteen patients were identified with high-risk resected melanoma. The median age at diagnosis was 8.5 years. The median time to establish diagnosis was 9 months. Primary lesions were diagnosed as unequivocal melanoma, atypical epithelioid melanocytic proliferations, or atypical Spitz tumor with indeterminate malignant potential. Twelve patients had a positive sentinel lymph node (SLN) biopsy or a palpable regional lymph node and underwent regional lymph node dissection (LND). Two patients with unequivocal melanoma with Breslow depth >4 mm had negative SLN biopsies. Twelve patients received adjuvant high-dose interferon. The following toxicities were observed: constitutional symptoms, gastrointestinal symptoms, depression or neuropsychiatric symptoms, myelosuppression, elevated AST or ALT, hypothyroidism, and hypertension. Grade 3 or 4 toxicities were uncommon with exception of neutropenia, resulting in modification of therapy in one patient. All patients are alive and free of disease at follow-up (median 24.5 months). Invasive melanoma can occur in very young children. Despite early signs of malignancy, there is often a delay in diagnosis. Histologically, diagnosis may be difficult because of overlap with Spitz nevi. Pediatric patients tolerated adjuvant high-dose interferon well and may be less likely than adults to require therapy modification secondary to toxicities.