Comprehensive Clinical, Histopathologic, and Molecular Analysis and Long-term Follow-up of Patients With Nodal Blue Nevi.

Blue nevi are benign, melanocytic neoplasms that show a range of clinical and morphologic patterns and include common/dendritic, cellular, and atypical cellular subtypes. Like other nevi, they most commonly occur in skin but can occasionally involve lymph nodes where they may be misinterpreted as representing metastatic melanoma. Moreover, whether benign blue nevi can metastasize to lymph nodes and their natural history and prognostic significance has been the subject of great controversy. To date, few cases of nodal blue nevi have been reported in the literature, and those reports have had limited clinical follow-up and supporting molecular data. This study sought to determine the clinical, pathologic, and molecular features of blue nevi involving lymph nodes, clarify their clinical significance, provide evidence for understanding their pathogenesis, and highlight potential pitfalls in the interpretation of lymph nodes with an ultimate aim of improving patient care. Thirteen cases of blue nevi involving lymph nodes were identified in the archives of Royal Prince Alfred Hospital, Sydney, Australia (1984-2018). A detailed assessment of the clinical and pathologic features of each case was performed, including an evaluation of all available immunohistochemical stains. Extended clinical follow-up was available for 9 patients. Droplet digital polymerase chain reaction for GNAQ Q209L, Q209P and GNA11 Q209L mutations was performed on 7 cases of blue nevi within lymph nodes together with matching cutaneous (presumed primary) blue nevi in 2 cases. All cases showed typical histologic features of blue nevi. BAP1 was retained in all cases (n=7). There were no recurrence or metastasis of blue nevus in any case on long-term clinical follow-up (n=9, median follow-up, 12 y). The majority of cases (n=5 of 7 evaluated) had GNAQ and GNA11 driver mutations. The 2 patients with a matched primary cutaneous blue nevus and regionally associated nodal blue nevus had the same GNAQ Q209L mutation in both sites in each patient. We conclude that blue nevi can involve lymph nodes and are associated with benign clinical behavior, and probably represent so-called "benign" metastasis. Awareness of these lesions is important when evaluating lymph nodes to avoid misdiagnosis as metastatic melanoma.

Molecular Profiling of Noncoding Mutations Distinguishes Nevoid Melanomas From Mitotically Active Nevi in Pregnancy.

The accurate recognition of subtle melanomas and their distinction from benign mimics is an oft-recurring diagnostic problem, critical for patient management. Melanomas that bear resemblance to benign nevi (so-called nevoid melanomas, NMs) and benign mitotically active nevi in pregnancy (MANP) are 2 lesions particularly prone to error. Molecular data, including analysis of noncoding regions, in MANP and NM are very limited. This study sought to identify differences in clinical, pathologic, and molecular characteristics between MANP and NMs to facilitate correct diagnosis and reduce the risk of overtreatment or undertreatment. Clinicopathologic characteristics of NM (n=18) and MANP (n=30) were evaluated, and mutation data were analyzed using next-generation sequencing for available cases in each group (NM, n=8; MANP, n=12). All MANP showed innocent histopathologic characteristics apart from increased mitotic activity, frequently in both superficial and deep parts of the lesion (median dermal mitotic rate: 2/mm, range: 1 to 7/mm). All cases of NM demonstrated a characteristic nevoid silhouette, subtle atypical architectural and cytologic features, and variable mitoses (median mitotic rate: 3/mm, range: 1 to 5/mm). Median NM tumor thickness was 1.4 mm. Four of 10 NM patients with follow-up had metastatic disease, including 3 patients who developed widespread metastases, with 1 disease-related death. No other recurrences have been identified (follow-up period: 24 to 60 mo). None of the 15 MANP patients with available follow-up had a recurrence. Most NMs harbored hotspot mutations in NRAS (6/8, 75%). Noncoding mutations were significantly more common in NMs than in MANP (median: 4 vs. 0, P=0.0014). Copy number alterations were infrequent but, when present, were seen in NMs (3/8 NMs vs. 0/12 MANP). All NMs but only 1 of 12 MANP had >1 abnormality in the noncoding regions. Similar to conventional common acquired nevi, MANP mostly harbored driver BRAF mutations, while activating NRAS mutations, noncoding mutations, and copy number alterations were rare. NM and MANP have subtle but recognizable distinguishing histopathologic characteristics that are underpinned by molecular differences. Mutation analysis of targeted noncoding mutations may assist in the diagnosis of difficult lesions.

FISH analysis of selected soft tissue tumors: Diagnostic experience in a tertiary center.

AIM: Fluorescence in situ hybridization (FISH) is an important ancillary tool for the classification of bone/soft tissue (BST) tumors. The aim of this study was to evaluate the contribution of FISH to the final classification of common BST entities in the molecular pathology department of the Royal Prince Alfred Hospital (RPAH), which is one of the most important referral centers for the management of sarcomas in Australia. METHODS: All routine diagnostic FISH tests performed on BST formalin-fixed paraffin embedded (FFPE) tissue specimens at the RPAH in a 5-year period (February, 2010-November, 2015) were reviewed. FISH analyses presented in this study include commercial break-apart probes (SS18, FUS, DDIT3, FUS, USP6, PDGFB, TFE3 and ALK) and a single enumeration (MDM2) probe. RESULTS: There were 434 interpretable FISH assays on BST samples including MDM2 (n=180), SS18 (n=97), FUS (n=64), DDIT3 (n=37), USP6 (n=30), PDGFB (n=13), TFE3 (n=8) and ALK (n=5). Discrepancies between the histopathological diagnosis and the FISH results were seen in 12% of the cases. In this subset of discordant cases, FISH contributed to the re-classification of 7% of cases originally diagnosed as synovial sarcoma (SS18) and 6% of adipocytic neoplasms (MDM2) based on the presence or absence of the expected gene alteration. CONCLUSION: Our study confirms that paraffin FISH is a sensitive and specific ancillary tool in the diagnosis of BST neoplasms when used in the appropriate clinicopathological context. These findings highlight the need for further ancillary molecular tools in the diagnosis and characterization of challenging cases.

Atypical Ewing sarcoma breakpoint region 1 fluorescence in-situ hybridization signal patterns in bone and soft tissue tumours: diagnostic experience with 135 cases.

AIMS: Recurrent Ewing sarcoma breakpoint region 1 (EWSR1) gene rearrangements characterize a select group of bone and soft tissue tumours. In our routine diagnostic practice with fluorescence in-situ hybridization (FISH), we have occasionally observed EWSR1 gene rearrangements in tumours not associated classically with EWSR1 translocations. This study aimed to review our institutional experience of this phenomenon and also to highlight the occurrence of unusual EWSR1 FISH signals (i.e. 5' centromeric region or 3' telomeric region signals) that do not fulfil the published diagnostic criteria for rearrangements. METHODS AND RESULTS: Using an EWSR1 break-apart probe, we performed FISH assays on formalin-fixed paraffin-embedded tissue sections from 135 bone and soft tissue specimens as part of their routine diagnostic work-up. EWSR1 gene rearrangements were identified in 51% of cases, 56% of which also showed an abnormal FISH signal pattern (in addition to classically rearranged signals). However, atypical FISH signals were present in 45% of the non-rearranged cases. In addition, we observed tumours unrelated to those described classically as EWSR1-associated that were technically EWSR1-rearranged in 6% of cases. Borderline levels of rearrangement (affecting 10-30% of lesional cells) were present in an additional 17% of these cases. CONCLUSIONS: While our study confirmed that FISH is a sensitive and specific tool in the diagnosis of EWSR1-associated tumours, atypical FISH signals and classical rearrangement in entities other than EWSR1-associated tumours can occur. Therefore, it is essential that the FISH result not be used as an isolated test, but must be evaluated in the context of clinical features, imaging, pathological and immunohistochemical findings.

The regenerating naevus.

The re-emergence of a melanocytic proliferation at the site of a previously excised pigmented lesion may not only cause great concern clinically but may also be amongst the most difficult of all melanocytic lesions for pathologists to assess. These lesions can adopt an appearance which may be impossible to confidently distinguish from a regressing or traumatised melanoma on histological grounds alone. For this reason, careful attention must be paid to the clinical context which has given rise to the lesion or a misdiagnosis may occur. In the absence of a corroborating history of prior surgery or trauma to the site, a diagnosis of a regenerating naevus may only be provisional. When considering a diagnosis of regenerating naevus, whenever possible, it is important to review and confirm the benign nature of the precursor lesion. Nevertheless, 50 years of research into this phenomenon has identified certain characteristic clinical features and histological patterns which provide clues both to clinicians and pathologists that will assist them to make the correct diagnosis and avoid over diagnosing as melanoma what is ultimately a benign process.

Punch biopsy of melanoma causing tumour cell implantation: another peril of utilising partial biopsies for melanocytic tumours.

The recommended initial management for suspected melanoma is excisional biopsy. The use of partial biopsies of melanocytic tumours poses potential problems including misdiagnosis due to either unrepresentative sampling or the difficulty in evaluating important diagnostic features; an inaccurate assessment of Breslow thickness and other important prognostic features; and the induction of changes capable of mimicking melanoma (i.e., pseudomelanoma). Misdiagnosis, in turn, may lead to inappropriate management of the patient and an adverse outcome. In this report we document a previously unrecognised pitfall of partial biopsies of melanocytic tumours: implantation of tumour cells at the biopsy site potentially leading to the overestimation of tumour thickness or a misdiagnosis of the presence of microsatellites in the subsequent wide excision specimen.

Digital papillary adenocarcinoma: a tumour that should be considered in the differential diagnosis of neoplasms involving the digits.

BACKGROUND: Digital papillary adenocarcinoma (DPA) is a rare malignant adnexal tumour that occurs almost exclusively in a digital location. Only two case series have been reported previously. AIMS: To document the clinical and histopathological findings in a series of patients with DPA. RESULTS: Of the 8 patients identified, six were males and two were females, and they were aged between 32 and 76 years at diagnosis. The diagnosis was not clinically suspected in any of the patients, and the tumours were thought to be cysts in four cases. The tumours ranged from 7 mm to 40 mm in size, and were located on the right thumb (n=3), the right middle finger (n = 3) and the left index finger (n = 2). Each tumour exhibited a multinodular growth pattern, with a combination of solid and cystic architecture. Papillary projections were present in many of the cystic spaces. In three cases, some cystic spaces were lined by luminal columnar epithelial cells and underlying myoepithelial cells. Cytological atypia was generally mild and mitotic activity was low in most cases (median 2 mitoses per mm). Local tumour recurrence occurred in three patients, of whom one subsequently had a digital amputation. No metastases have been identified in the seven patients with known follow-up information. CONCLUSIONS: DPA is a rare tumour that should be included in the differential diagnosis of epithelial neoplasms in digital locations. Because of its tendency to recur locally, wide local excision should be performed as definitive initial treatment.

Tumor-infiltrating lymphocyte grade is an independent predictor of sentinel lymph node status and survival in patients with cutaneous melanoma.

PURPOSE: To determine whether density and distribution of tumor-infiltrating lymphocytes (TILs; TIL grade) is an independent predictor of sentinel lymph node (SLN) status and survival in patients with clinically localized primary cutaneous melanoma. METHODS: From the Melanoma Institute Australia database, 1,865 patients with a single primary melanoma ≥ 0.75 mm in thickness were identified. The associations of clinical and pathologic factors with SLN status, recurrence-free survival (RFS), and melanoma-specific survival (MSS) were analyzed. RESULTS: The majority of patients had either no (TIL grade 0; 35.4%) or few (TIL grade 1; 45.1%) TILs, with a minority showing moderate (TIL grade 2; 16.3%) or marked (TIL grade 3; 3.2%) TILs. Tumor thickness, mitotic rate, and Clark level were inversely correlated with TIL grade (each P < .001). SLN biopsy was performed in 1,138 patients (61.0%) and was positive in 252 (22.1%). There was a significant inverse association between SLN status and TIL grade (SLN positivity rates for each TIL grade: 0, 27.8%; 1, 20.1%; 2, 18.3%; 3, 5.6%; P < .001). Predictors of SLN positivity were decreasing age (P < .001), decreasing TIL grade (P < .001), ulceration (P = .003), increasing tumor thickness (P = .01), satellitosis (P = .03), and increasing mitoses (P = .03). The 5-year MSS and RFS rates were 83% and 76%, respectively (median follow-up, 43 months). Tumor thickness (P < .001), ulceration (P < .001), satellitosis (P < .001), mitotic rate (P = .003), TIL grade (P < .001), and sex (P = .01) were independent predictors of MSS. Patients with TIL grade 3 tumors had 100% survival. CONCLUSION: TIL grade is an independent predictor of survival and SLN status in patients with melanoma. Patients with a pronounced TIL infiltrate have an excellent prognosis.

Sentinel lymph nodes containing very small (<0.1 mm) deposits of metastatic melanoma cannot be safely regarded as tumor-negative.

BACKGROUND: Some authors have suggested that patients with very small (<0.1 mm) deposits of metastatic melanoma in sentinel lymph nodes (SLNs) should be considered SLN-negative, whereas others have reported that such patients can have adverse long-term outcomes. The aims of the present study were to determine whether extensive sectioning of SLNs resulted in more accurate categorization of histologic features of tumor deposits and to assess prognostic associations of histologic parameters obtained using more intensive sectioning protocols. METHODS: From patients with a single primary cutaneous melanoma who underwent SLN biopsy between 1991 and 2008, those in which the maximum size of the largest tumor deposit (MaxSize) in SLNs was <0.1 mm in the original sections were identified. Five batches of additional sections were cut from the SLN tissue blocks at intervals of 250 µm. The 1st batch was cut from the blocks without any trimming; these sections were therefore immediately adjacent to the original sections. Each batch included 5 sequential sections, the 1st and 5th stained with hematoxylin-eosin, and the 2nd, 3rd, and 4th stained immunohistochemically with S-100, HMB-45, and Melan-A, respectively. In each batch of sections, the following histologic features of tumor deposit(s) in the SLNs were evaluated: MaxSize; tumor penetrative depth (TPD) (defined as the maximum depth of tumor deposit(s) from the inner margin of the lymph node capsule), and intranodal location (classified as subcapsular if the tumor deposit(s) were confined to the subcapsular zone or parenchymal if there was any involvement of the nodal parenchyma beyond the subcapsular zone). The measured histologic parameters were compared in each batch of sections. The association of histologic parameters with overall survival was assessed for the parameters measured in each batch of sections. RESULTS: There were 20 eligible patients (15 females, 5 males, median age 60 years). After a median follow-up duration of 40 months, 4 patients had died from melanoma and 2 patients of unknown causes. Completion lymph node dissection (CLND) was performed in 13 cases (65%) and was negative in all cases. Relative to the measured values on the original sections, all 3 parameters were upstaged in subsequent batches of sections, but no further upstaging of MaxSize, TPD, or location was seen beyond batch 3, batch 4, and batch 2, respectively. Increasing MaxSize was associated with significantly poorer overall survival in batches 1, 2, and 3. Parenchymal involvement was significantly associated with poorer survival in batches 2-5. TPD was not significantly associated with overall survival. CONCLUSIONS: The results of this study indicate that very small (<0.1 mm) deposits of melanoma in SLNs may be associated with adverse clinical outcomes and that this is due, at least in part, to the underestimation of SLN tumor burden in the initial sections. Our evidence does not support clinical decision-making on the assumption that patients with very small melanoma deposits in SLNs have the same outcome as those who are SLN-negative.

Clinical and pathological features of metastases of primary cutaneous desmoplastic melanoma.

AIMS: Primary cutaneous desmoplastic melanoma (DM) may be entirely desmoplastic ['pure' DM (pDM)] or exhibit a desmoplastic component admixed with a non-desmoplastic component ['combined' DM (cDM)]. Our aim was to describe the histological features of metastases of primary DM and to determine whether they were predictive of outcome. METHODS: The effect of clinicopathological parameters on overall survival (OS) was analysed, and the correlation between histological features of the primary melanoma and metastases was studied in patients with metastatic DM. RESULTS: Twenty-six patients (18 males; eight females) developed 50 metastases in sentinel nodes (13; 26.0%), regional nodes (10; 20.0%), skin (14; 28.0%), and distant sites (13; 26.0%). The cellular composition of metastases correlated with that of the corresponding primary tumours. Time to development of first non-sentinel lymph node metastasis was shorter in cDM than in pDM (median 11.2 versus 24.9 months, P = 0.075). The only independent predictors of poorer OS were cDM type [hazard ratio 6.17, 95% confidence interval (CI) 1.61-23.81, P = 0.008] and male sex (hazard ratio 5.98, 95% CI 1.34-26.66, P = 0.019). CONCLUSIONS: The cellular composition of primary DM correlates with that of metastatic DM and with outcome. It is important to consider the possibility of primary or metastatic DM when examining tumours composed of spindle cells.