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Utility of next generation sequencing for differentiating malignant PEComa from metastatic melanoma

  • Elizabeth Paver
    Affiliations
    Melanoma Institute Australia, The University of Sydney, Sydney, NSW, Australia

    NSW Health Pathology, Sydney, NSW, Australia

    Royal Prince Alfred Hospital, Sydney, NSW, Australia
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  • Karina Aivazian
    Affiliations
    Melanoma Institute Australia, The University of Sydney, Sydney, NSW, Australia

    NSW Health Pathology, Sydney, NSW, Australia

    Royal Prince Alfred Hospital, Sydney, NSW, Australia
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  • Fiona Maclean
    Affiliations
    Douglass Hanly Moir Pathology, Sonic Healthcare, Macquarie Park, NSW, Australia

    Department of Clinical Medicine, Faculty of Medicine and Health Sciences, Macquarie University, Sydney, NSW, Australia
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  • Georgina V. Long
    Affiliations
    Melanoma Institute Australia, The University of Sydney, Sydney, NSW, Australia

    Charles Perkins Centre, The University of Sydney, Sydney, NSW, Australia

    Royal North Shore and Mater Hospitals, Sydney, New South Wales, Australia

    Faculty of Medicine and Health, The University of Sydney, NSW, Australia
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  • Andrew J. Spillane
    Affiliations
    Melanoma Institute Australia, The University of Sydney, Sydney, NSW, Australia

    Royal North Shore and Mater Hospitals, Sydney, New South Wales, Australia

    Faculty of Medicine and Health, The University of Sydney, NSW, Australia
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  • James S. Wilmott
    Affiliations
    Melanoma Institute Australia, The University of Sydney, Sydney, NSW, Australia

    Charles Perkins Centre, The University of Sydney, Sydney, NSW, Australia

    Faculty of Medicine and Health, The University of Sydney, NSW, Australia
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  • Richard A. Scolyer
    Correspondence
    Contact: Prof Richard A. Scolyer.
    Affiliations
    Melanoma Institute Australia, The University of Sydney, Sydney, NSW, Australia

    NSW Health Pathology, Sydney, NSW, Australia

    Royal Prince Alfred Hospital, Sydney, NSW, Australia

    Charles Perkins Centre, The University of Sydney, Sydney, NSW, Australia

    Faculty of Medicine and Health, The University of Sydney, NSW, Australia
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Published:November 16, 2022DOI:https://doi.org/10.1016/j.pathol.2022.08.011
      To the Editor,
      Differentiating between metastatic melanoma and malignant perivascular epithelioid cell tumour (malignant PEComa; also known as malignant epithelioid angiomyolipoma) may present a difficult diagnostic challenge due to the presence of overlapping histological and immunohistochemical features, particularly when occurring in patients with a history of melanoma. Given the marked differences in their natural history, prognosis and management (including systemic therapy options), distinguishing between them is critical for optimal patient care. Herein, we present a case of a high grade epithelioid malignancy associated with a conventional angiomyolipoma/PEComa of the kidney, in a patient with a history of melanoma, in which the ultimate diagnosis was established by analysing the primary melanoma and the two distinct areas of the kidney tumour using a next generation sequencing (NGS) panel. The case highlights the utility of DNA sequencing to establish the correct diagnosis when the results of routine clinical, morphological, immunohistochemical and molecular evaluation remain inconclusive.
      A 58-year-old female presented with abdominal pain. An abdominal positron emission tomography-computed tomography (PET-CT) scan identified a biphasic, PET-avid left renal mass. No other suspicious lesions were apparent on clinical examination or imaging. She had a history of primary cutaneous melanoma of the thigh resected 3 years previously, which showed high risk features including a Breslow thickness of 10 mm and an elevated mitotic rate (5 mitoses/mm2), although no metastatic disease was identified in three sentinel lymph nodes from the right inguinal and iliac crest regions at that time. The melanoma was predominantly intradermal, with a growth pattern reminiscent of a deep penetrating naevus but with atypical spindled cytomorphology, prominent nuclear pleomorphism and frequent mitotic activity (Fig. 1A–D). It was positive with melanocyte markers (SOX10, S100, Melan-A and HMB45) and PRAME, while p16 expression was lost. It was negative for desmin, CD31, ERG, CD10, cytokeratin and p63.
      Fig. 1
      Fig. 1(A–D) Primary melanoma on the right thigh. (A) Low power image of the melanoma extending into the deep dermis. (B) Melanoma infiltrating between dermal collagen. (C) Atypical melanocytes with pale eosinophilic cytoplasm and focal melanin pigment. (D) High power image showing cytological atypia, spindled and epithelioid morphology with abundant pale cytoplasm and mitoses. Inset: the melanoma was positive with PRAME and negative with p16. It was also positive with S100, SOX10, HMB45 and Melan-A (not shown). (E–K) Renal tumour. (E) Macroscopic image of exophytic, solid tumour at upper pole; with variegated cut surface and discrete nodule, with focal areas of possible haemorrhage and necrosis. (F,G) Low grade region showing characteristic features of angiomyolipoma including myoid spindle cells, vessels and fat. (H,I) Interface between regions, with abrupt transition in some areas (H) and a more intermingled appearance elsewhere (I). (J,K) High grade region, composed of epithelioid cells showing areas of perivascular condensation (J), and pseudo-alveolar growth with frequent mitoses (red circles) (K).
      The left renal mass was removed with a radical nephrectomy. No extrarenal disease was identified intraoperatively. On macroscopic pathological examination, the upper pole of the kidney contained an exophytic, 44×35×23 mm tumour with a mainly solid, lobulated, focally nodular appearance. The cut surface was heterogeneous, with cream, pale tan and brown areas, with focal necrosis and haemorrhage (Fig. 1E). Histologically, the tumour had a biphasic appearance, with one region showing features of a classical angiomyolipoma/PEComa, and the other showing overt features of malignancy. The two regions were discretely separated in some areas, and more intimately admixed elsewhere (Fig. 1H,I). The low grade region (Fig. 1F,G) contained bland spindle cells with myoid features, some with cytoplasmic clearing, arranged in short fascicles. Focal adipocytes and variably sized vessels were identified. No mitoses or necrosis were seen in this area. The second component consisted of large, epithelioid cells with eosinophilic cytoplasm and prominent nucleoli, showing solid and pseudopapillary growth, with some areas showing perivascular condensation, areas of necrosis, and up to 18 mitoses per 10 high power fields (Fig. 1J,K).
      The immunohistochemical findings are summarised in Fig. 2. The low grade region of the kidney tumour showed diffuse positivity with SMA, patchy staining with desmin and HMB45, and focal weak positivity with Melan-A (Fig. 3). It was negative for S100, SOX10, PRAME and p16. There was focal, weak cytoplasmic positivity for WT1, patchy positivity for CD56, and occasional cells were positive for CCNB3. The high grade region showed patchy staining for S100 and moderate staining for PRAME and Cyclin D1, with focal HMB45 positivity and focal weak Melan-A positivity (Fig. 3). SOX10 was negative across multiple blocks of the high grade component. CD56 and CCNB3 were positive, WT1 showed cytoplasmic positivity, there was non-specific cytokeratin uptake in the necrotic areas, and p16 showed patchy positive staining. BRAFVE1, TFE3, GATA3, CD57, MyoD1 and Myogenin were negative in both components, and INI1 was retained. In both components MiTF was weakly positive.
      Fig. 3
      Fig. 3Immunohistochemical profile of the two regions in the kidney tumour. (A,B) HMB45 showing patchy positivity in both low grade (A) and high grade (B) regions. (C) Melan-A showed very focal weak staining in both regions; image from interface between zones. (D,E) Smooth muscle actin (SMA) stain, with the intermingled interface (D) and the abrupt interface (E) between zones. SMA showed diffuse positivity in the spindled, low grade region (left half of each image) and focal, very weak positivity in high grade region (right half). (F) S100 showed focal positivity in the high grade region (right half of image). (G) CD56 showed patchy positivity in both regions. (H) PRAME showed nuclear positivity in the high grade region (top right of image). (I) WT1 showed diffuse cytoplasmic/membrane staining in the high grade region (right half of image) and focal positivity in low grade region.
      Differential diagnoses based on the morphology and immunoprofile included an angiomyolipoma/PEComa with malignant transformation, or a benign angiomyolipoma involved by metastatic melanoma. Initially it was considered that, despite the negativity with SOX10, the malignant component was morphologically more in keeping with metastatic melanoma than a malignant PEComa. Routine molecular analysis for metastatic melanoma was performed on the primary melanoma and the kidney tumour at that time using a PCR-based DNA sequencing assay (Illumina technology), which showed no hotspot mutations in BRAF, NRAS or KIT in either tumour. The key pathological features are summarised in Fig. 2.
      In light of the prognostic and treatment implications of the two differential diagnoses, additional DNA sequencing was performed on tissue from the patient's primary melanoma and separately on each component of the renal tumour using a customised next generation sequencing panel (ArcherDX), which incorporated a total of 54 recurrently mutated coding and non-coding melanoma-related genes discovered in our previous whole genome sequencing of a large cohort of melanomas.
      • Hayward N.K.
      • Wilmott J.S.
      • Waddell N.
      • et al.
      Whole-genome landscapes of major melanoma subtypes.
      This revealed abnormalities in the primary melanoma that were absent in both zones of the kidney lesion, including a CDKN2A mutation and a TERT promoter mutation. In contrast, the two components of the kidney tumour harboured an identical TP53 mutation, which was absent from the melanoma (Fig. 2). These findings indicated that the malignant renal tumour was molecularly more similar to the adjacent angiomyolipoma/PEComa than to the melanoma. Consequently, the kidney tumour was diagnosed as a malignant PEComa arising in association with an angiomyolipoma/PEComa. Within 12 months of diagnosis the patient developed metastatic PEComa in retroperitoneal lymph nodes, as well as a cerebellar dysfunction syndrome that was considered to be paraneoplastic in nature.
      Angiomyolipoma, a member of the family of perivascular epithelioid cell neoplasms (PEComa), represents a rare mesenchymal neoplasm with an unusual immunoprofile, expressing both smooth muscle markers (SMA, desmin and caldesmon) and melanocytic markers (usually Melan-A and HMB45 and sometimes S100).
      WHO Classification of Tumours Editorial Board
      WHO Classification of Tumours: Soft Tissue and Bone Tumours.
      Most cases are sporadic, however a subset arises in association with tuberous sclerosis complex.
      • Rakowski S.K.
      • Winterkorn E.B.
      • Paul E.
      • et al.
      Renal manifestations of tuberous sclerosis complex: incidence, prognosis, and predictive factors.
      ‘Classical’ angiomyolipoma is considered benign, and has characteristic histological features that allow diagnosis on routine histology. However, the epithelioid variant has malignant potential and can occasionally demonstrate histological features similar to melanoma including large polygonal or spindled cells with atypical nuclei, prominent nucleoli, intranuclear inclusions, multinucleated giant cells, mitoses and necrosis.
      • Holger M.
      • Humphrey P.A.
      • Ulbright T.M.
      • et al.
      WHO Classification of Tumours of the Urinary System and Male Genital Organs.
      While it is not often that malignant PEComa of the kidney occurs in a patient with a history of melanoma, distinguishing between these entities is of significant clinical importance due to the implications for both prognosis and ongoing management. A diagnosis of resected American Joint Committee on Cancer (AJCC) stage IV metastatic melanoma would result in consideration of adjuvant immune checkpoint inhibitor therapy, and possible enrolment into a clinical trial. Malignant PEComa often shows resistance to chemotherapy and radiotherapy, and thus the mainstay of treatment is radical resection and close observation, with long-term follow-up due to the risk of late recurrence.
      • Sobiborowicz A.
      • Switaj T.
      • Teterycz P.
      • et al.
      Feasibility and long-term efficacy of PEComa treatment-20 years of experience.
      Immunohistochemistry does not necessarily help differentiate these entities. Melan-A and HMB45, markers that are otherwise considered relatively specific for melanocyte differentiation, are positive in angiomyolipoma/PEComa. While SOX10 and S100 positivity would favour a diagnosis of melanoma, absence of staining does not exclude this entity: the capacity of melanoma to dedifferentiate and lose expression of one or more melanocyte markers is well known.
      • Agaimy A.
      • Stoehr R.
      • Hornung A.
      • et al.
      Dedifferentiated and undifferentiated melanomas: report of 35 new cases with literature review and proposal of diagnostic criteria.
      In addition, while expression of smooth muscle markers may be more suggestive of a PEComa, melanoma may also show divergent differentiation, aberrantly expressing other markers (including muscle markers).
      • Agaimy A.
      • Stoehr R.
      • Hornung A.
      • et al.
      Dedifferentiated and undifferentiated melanomas: report of 35 new cases with literature review and proposal of diagnostic criteria.
      Routine melanoma-related molecular testing may be useful if a common melanoma-associated driver mutation (such as a BRAF or NRAS mutation) is identified, however almost 50% of melanomas are negative for these mutations, thus, if absent/wild-type, melanoma still remains a possibility.
      • Lyle M.
      • Haydu L.E.
      • Menzies A.M.
      • et al.
      The molecular profile of metastatic melanoma in Australia.
      A subset of PEComas harbour translocations in TFE3,
      WHO Classification of Tumours Editorial Board
      WHO Classification of Tumours: Soft Tissue and Bone Tumours.
      which can be identified using immunohistochemistry and with fluorescent in situ hybridisation (FISH). If positive, this may assist in recognising a PEComa, however it was negative in our case and thus did not assist in the diagnosis.
      The most common molecular event in angiomyolipoma/PEComa is deletion and/or loss of heterozygosity in TSC2 or TSC1; genes which were not included in our customised melanoma NGS panel.
      • Agaram N.P.
      • Sung Y.S.
      • Zhang L.
      • et al.
      Dichotomy of genetic abnormalities in PEComas with therapeutic implications.
      A subgroup of PEComa is characterised by rearrangements of the TFE3 gene, an abnormality that is mutually exclusive with TSC2 mutations.
      • Agaram N.P.
      • Sung Y.S.
      • Zhang L.
      • et al.
      Dichotomy of genetic abnormalities in PEComas with therapeutic implications.
      TERT promoter mutations occur in about 80% of cutaneous melanomas,
      • Hayward N.K.
      • Wilmott J.S.
      • Waddell N.
      • et al.
      Whole-genome landscapes of major melanoma subtypes.
      but have not, to our knowledge, been identified in PEComa. Loss of function of cyclin-dependent kinase inhibitor 2A (CDKN2A), also a frequent event in primary invasive melanoma,
      • Hayward N.K.
      • Wilmott J.S.
      • Waddell N.
      • et al.
      Whole-genome landscapes of major melanoma subtypes.
      appears to be an uncommon occurrence in PEComa.
      • Groisberg R.
      • Hong D.S.
      • Holla V.
      • et al.
      Clinical genomic profiling to identify actionable alterations for investigational therapies in patients with diverse sarcomas.
      On the other hand, mutations in TP53 are a recognised event in many PEComas
      • Groisberg R.
      • Hong D.S.
      • Holla V.
      • et al.
      Clinical genomic profiling to identify actionable alterations for investigational therapies in patients with diverse sarcomas.
      ,
      • Groisberg R.
      • Subbiah V.
      Sequencing PEComas: viewing unicorns through the molecular looking glass.
      but are relatively rare in melanoma.
      • Hodis E.
      • Watson I.R.
      • Kryukov G.V.
      • et al.
      A landscape of driver mutations in melanoma.
      The NGS results thus provided the evidence needed to demonstrate the molecular similarity between the malignant renal component and the classical angiomyolipoma, and exclude metastatic melanoma.
      This case highlights the utility of deep sequencing molecular analysis to establish a correct diagnosis in cases where results of clinical, morphological, immunohistochemical and standard molecular evaluation remain inconclusive, particularly where the findings will have an immediate impact on patient management.

      Conflicts of interest and sources of funding

      RAS has received fees for professional services from F. Hoffmann-La Roche Ltd, Evaxion, Provectus Biopharmaceuticals Australia, Qbiotics, Novartis, Merck Sharp & Dohme, NeraCare, AMGEN Inc., Bristol-Myers Squibb, Myriad Genetics, GlaxoSmithKline. GVL is consultant advisor for Agenus, Amgen, Array Biopharma, Boehringer Ingelheim, Bristol Myers Squibb, Evaxion, Hexal AG (Sandoz Company), Highlight Therapeutics S.L., Merck Sharpe & Dohme, Merck Sharpe & Dohme, Novartis, OncoSec, Pierre Fabre, Provectus, Qbiotics, Regeneron.
      This work was supported by a National Health and Medical Research Council of Australia (NHMRC) Program Grant ( APP1093017 ) (to RAS and GVL). RAS is supported by an NHMRC Practitioner Fellowship ( APP1141295 ). GVL is supported by an NHMRC Investigator Grant and the University of Sydney Medical Foundation. Support from the BB and A Miller Foundation (the Jani Haenke Melanoma Pathology Fellowship to ECP), The Ainsworth Foundation, and the CLEARbridge Foundation, as well as from colleagues at Melanoma Institute Australia, Royal Prince Alfred Hospital, and NSW Health Pathology, is also gratefully acknowledged. The authors state that there are no other conflicts of interest to disclose.

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