June 25, 2019 Join the Discussion at #Asceconf

2019-2020 SERIES HANDOUT

June 25, 2019

Challenges and Pitfalls in Cytomorphology of Melanoma

Xiaoqi Lin, MD, PhD

Join the discussion at #asceconf

100 West 10th Street, Suite 605 Wilmington, DE 19801 Phone: (302) 543-6583 ext 103 | Fax: (302) 543-6597 Email: [email protected] Web site: cytopathology.org

6/20/2019

ASC Cytoeconference Challenges and Pitfalls in Cytology of Malignant Melanoma

Xiaoqi Lin, MD, PhD Northwestern University Chicago, IL

Dr. Lin has no relevant financial and ethical conflicts to report.

Educational Objectives 1. Utilize a case based approach to highlight the various cytomorphologic features of melanoma in FNA smears and touch prep of cores. 2. Discuss pitfalls and differential diagnosis of melanoma cytology. 3. Discuss updated ancillary studies for melanoma.

Malignant Melanoma (MM)  Melanoblasts are neural crest derivatives and are notorious for causing primary cutaneous neoplasms.  Accounts for 1 - 3% of all malignancies.  The most common primary site is skin (foot followed by maxilla and thigh).  Any organ system may be involved.  Metastatic melanoma of unknown origin accounts for 2 - 6% of all melanoma cases.

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Sampling Methodology  Fine needle aspiration (FNA): Percutaneous, EUS, transcleral and vitrectomy-assisted transvitreal.  Core needle biopsy (CNB) with touch preparation (TP): Percutaneous, EBUS and EUS.  Body fluids.  Pap smears

LN Staging of MM Patients  US: Is a method for the early detection of clinically nonevidential metastases. Sensitivity: 25 - 50%. Specificity: 81%. Gamma probe- guided US is a highly accurate method for correctly identifying the sentinel LN.  FNA: Should be performed early, which provides a diagnosis before identification of the primary lesion in up to 20% of cases. Sensitivity: 96.3%; Specificity: 98.9%. FNACs guided by palpation has sensitivity and specificity similar to that by US/gamma probe US (98.4% vs 97.2%, 100% vs 99.8%). It is still debated if US-FNAC adds significantly to the staging and management of patients with mainly thin cutaneous melanomas.  For the present, sentinel lymph node biopsy remains the most accurate method of regional lymph node staging in patients with newly diagnosed melanoma. 5

27y, M H/o melanoma in the ankle Palpable groin LN

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Discohesive Pattern

Differential Diagnosis  Benign lymph node  Burkitt lymphoma  Metastatic undifferentiated/ poorly differentiated carcinoma  Metastatic melanoma

Differential Diagnosis

Benign Lymph Node

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Differential Diagnosis

Burkitt Lymphoma

CD20

Differential Diagnosis

Undifferentiated/Poorly Differentiated Carcinoma

FISH – BRD3-NUT

S100

HMB45 Melan A

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Loosely Cohesive Clusters 3D-Dimentional Cohesive Clusters

Differential Diagnosis  Neuroendocrine neoplasm  Poorly differentiated / undifferentiated carcinoma  Malignant melanoma

Differential Diagnosis Neuroendocrine Neoplasm

Chromogranin

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Differential Diagnosis Poorly Differentiated/Undifferentiated Adenocarcinoma

Metastatic pancreatic adenocarcinoma to left supraclavicular lymph node 16

81y, M H/o smoking and prostate carcinoma Presents with hilar and mediastinal lymphadenopathy Biopsy a 2.4cm left paratracheal node

Pseudopapillary Pattern

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Differential Diagnosis  Metastatic solitary pseudopapillary tumor  Metastatic melanoma

Differential Diagnosis Solitary Pseudopapillary Tumor

-catenin

S100

HMB45 Melan A

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65y, M H/o colonic adenocarcinoma and melanoma Presents with a rib lesion

Plasmacytoid Cells with Binucleation / Multinucleation and Granular Cytoplasm

Intranuclear Cytoplasmic Invaginations

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Differential Diagnosis  Neuroendocrine neoplasm  Multiple myeloma / plasmacytoma  Melanoma

Differential Diagnosis Multiple myeloma / plasmacytoma

Kappa Lambda

Melan A

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57y, M H/o melanoma at R mid back (excision showed melanoma in situ and scar), and marginal cell lymphoma 3 cm PET avid R axillary LN

Spindle Cells

Differential Diagnosis  Sarcoma, including malignant peripheral nerve sheath tumor  Metastatic sarcomatoid carcinoma  Metastatic melanoma

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Differential Diagnosis Sarcoma, Including MPNST

Spindle Melanoma vs. MPNST  Markedly pleomorphic spindle cells forming cohesive fascicles or whorls intermingled, with individual cell apoptosis, necrosis and myxoid stroma.  Spindle melanoma: Scattered epithelioid tumor cells.  IHC: Positive for SOX10, vimentin and S-100 (Diffuse vs. focal). Negative for pan-CK. Melanoma tends to lose expression of HMB45 and Melan A.  EM: Melanoma - Intracytoplasmic melanosomes and cisternae of rough endoplasmic reticulum with intracisternal parallel tubules

Differential Diagnosis Sarcomatoid Carcinoma HCC Lung RCC Thyroid

AE1/AE3 Vimentin Vimentin AE1/AE3

HepPar-1 TTF1 CA IX PAX8

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SOX10 Desmin

MDM2

44y, F Presented as heel ulcerated lesion and enlarged inguinal LNs

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Lymphoma-Like Cells

Differential Diagnosis  Undifferentiated carcinoma  Lymphoma  PNET / Ewing’s sarcoma  Melanoma

Differential Diagnosis

Diffuse Large Cell Lymphoma

Positive for B cell markers (IHC, flow cytometry, ISH)

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Differential Diagnosis PNET / Ewing’s Sarcoma

CD99 40

HMB45 Melan A

CD3 CD20

67y, M H/o melanoma and prostatic adenocarcinoma, 3+3 and 3+4 Sample a 2 cm RLL nodule.

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Epithelioid / Spindle Cells with Pigment, Pigmented Macrophages, Necrosis

Differential Diagnosis  Anthracotic pigment  Hemosiderin pigment  Melanin pigment  Other sites  Lipofuscin pigment  Tattoo pigment

Differential Diagnosis Anthracotic Pigment

77y, M, with a history of melanoma; now with lung and kidney masses; sampling a 1.5 cm lung mass that was hot on PET; clinical suspicion is for metastatic disease. Reactive type II pneumocytes and macrophages

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Differential Diagnosis

Hemosiderin Pigment

Differential Diagnosis Lipofuscin Pigment

Thyroid Liver

Differential Diagnosis

Tattoo

SOX10

33y, F, h/o multiple melanomas, undergoing axillary LN bx. <1 cm. Pt. obtained a tattoo in the area of sample 8 years ago.

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S100 Melan A

HMB45 PSA TTF1

64y, F Biopsy a thigh lesion

Cells with Cytoplasmic Vacuolation

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Melan A HMB45

41y, M Back lesion 2.5 cm L axillary LN

Ballooning Cells

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Differential Diagnosis  Macrophages  Granular cell tumor  Melanoma

Differential Diagnosis

Macrophages

Differential Diagnosis Granular Cell Tumor

S100 Positive for SOX10; Negative for HMB45 and Melan A. 57

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S100

Melan A HMB45 CD68

44y, F Presented as heel ulcerated lesion Enlarged inguinal LNs

Small Cells

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Melanoma with Osteoclast-Like Giant Cells  May mean a more aggressive behavior.  Immunophenotype: +ve for CD68, MiTF, p16; -ve for S100, HMB45, Melan-A, SOX10, Ki67, CD163, BRAF. Melanoma with Cartilaginous Differentiation Melanoma with Rhabdoid Features  Enlarged, eccentrically placed nuclei with prominent nucleoli.  Moderate amount of cytoplasm possessing round, globular inclusions.  IHC: +ve for S-100, HMB-45 and vimentin.

Desmoplastic Melanoma  Less cellular.  Less often exhibited intranuclear cytoplasmic invaginations.  Less mitotic figures. 61

Melanoma in Body Fluid  More epithelioid feature.  Intranuclear cytoplasmic invaginations: 21.9%, less.  Melanin pigment: 25.0%.

Metastatic Melanoma in Pleural Fluid

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Metastatic Melanoma in Peritoneal Fluid

Metastatic Melanoma in CSF

S100

HMB45 Melan A

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Uveal Melanoma  Spindle cells: 98% (63% mixed and 35% spindle only).  Only epithelioid cells: 2%.  Melanin granules were observed in 80% of samples.  Iris melanoma has bland features compared with ciliary and choroidal melanoma.

SOX10

S100 Melan A 69

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Pap Smear

Excision of Malignant Melanoma

Classic Cytologic Features of Melanoma  Discohesive cellular pattern (94%) (loosely cohesive pattern)  Plasmacytoid morphology (78 - 88%) and spindle cells (88%)  Intranuclear pseudoinclusions (63 – 100%)  Macronucleoli / prominent nucleoli  Binucleation (94 – 100%)/ multinucleation (44%)  Cytoplasmic melanin pigments (44 – 77%), pigmented melanophages (88%)  Cytoplasmic vacuolation (83 – 88%)

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Less Common or Significant Cytologic Features of MM  Pseudopapillary pattern (19%)  Pure spindle cell dominant (6%)  Lymphoma-Like (6%)  Balloon cells  Necrosis (56%)

Ancillary Studies  IHC  Molecular studies

 Cell blocks  Cores  Direct-smear / cytospin / liquid-based slides

Immunohistochemistry of MM Immunomarkers Positivity SOX10 100% S100 87 - 95% Melan A/MART-1 88% HMB45 81% MiTF 83 – 90% KBA.62 75% COX-2 100% CD117 46% Pan-CK Negative Desmin Negative CD45 Negative Glypican-2 Negative 75

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Expression of SOX10 in Neurogenic Neoplasms

Neoplasms SOX10 Neoplasms SOX10 Benign PNST 100% Nerve sheath myxoma 100% Cellular blue nevus 100% Neuroblastoma 62% Clear cell sarcoma 95% Neurofibroma 100% Granular cell tumor, S100+ 100% Neurothekeoma 0% Granular cell tumor, S100- 0% Olfactory neuroblastoma 71% MPNST 22% Paraganglioma 75% Melanoma, metastatic 95% Perineurioma 0% Melanoma, desmoplastic 96% Schwannoma, soft tissue 99% Melanoma, sinonasal 88% Schwannoma, GI-tract 100%

Miettnen M, et al. Am J Surg Pathol. 2015 39:826-35. 76

Expression of SOX10 in Soft Tissue Neoplasms Neoplasm SOX10 Ossifying fibromyxoid tumor 4.3% Rhabdomyosarcoma, alveolar 7.4%

Neoplasms negative for SOX10: Alveolar soft part sarcoma, angiosarcoma, benign fibrous histiocytoma, chondrosarcoma, dermatofibrosarcoma protuberans, desmoid fibromatosis, desmoplastic small round cell tumor, epithelioid hemangioendothelioma, epithelioid sarcoma, Ewing sarcoma, fibrous hamartoma of infancy gastrointestinal stroma tumor, glomus tumor, glomangiopericytoma, hemangioma, inflammatory myofibroblastic tumor, Kaposi sarcoma, leiomyoma, leiomyosarcoma, liposarcoma, low-grade fibromyxoid sarcoma, meningioma, myofibroma, nodular fasciitis, perivascular epithelioid tumor (PEComa), pleomorphic undifferentiated sarcoma, proliferative fasciitis, rhabomyosarcoma-embryonal, sinonasal low-grade polyphenotypic sarcoma, solitary fibrous tumor/HPC), and synovial sarcoma.

Miettnen M, et al. Am J Surg Pathol. 2015 39:826-35. 77

Expression of SOX10 in Epithelial Neoplasms

Neoplasms SOX10 Neoplasms SOX10

Pleomorphic adenoma 99% Ductal carcinoma of salivary gland 6%

Adenoid cystic carcinoma 97% Pulmonary small cell carcinoma 7%

Breast ductal carcinoma 12% Squamous cell carcinoma of head and neck 6%

Ectopic hamartomatous thymoma 29% Squamous cell carcinoma of lung 3%

Embryonal carcinoma 35% Cylindroma/spiradenoma 100%

Pulmonary adenocarcinoma 1% Hidradenoma 9%

Mixed tumor/myoepithelioma 82% Malignant mixed tumor/myoepithelioma 30%

Neoplasms negative for SOX10: Parathyroid adenoma, adrenocortical carcinoma, basal cell carcinoma of skin, breast lobular carcinoma, chordoma, colorectal adenocarcinoma, endometrial adenocarcinoma, seminoma, choriocarcinoma, cholangiocarcinoma, hepatocellular carcinoma, mesothelioma, Merkel cell carcinoma, ovarian endometrioid carcinoma, ovarian sercous carcinoma, pancreatic adenocarcinoma, pancreatic neuroendocrine tumor, prostatic adenocarcinoma, renal cell carcinoma, renal oncocytoma, squamous cell carcinoma of skin, gastric adenocarcinoma, thymoma, thyroid carcinoma and urothelial carcinoma. Miettnen M, et al. Am J Surg Pathol. 2015 39:826-35. 78

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Expression of HMB45 and Melan A

Neoplasms HMB45 Melan A Melanoma /melanocytic nevus + + PEComas + + Clear cell sarcoma of soft tissue + + t(6;11)(p21;q12) renal cell carcinoma + + Pigmented neural tumor and melanotic schwannoma + + Pheochromocytoma (30%) + Epithelioid sarcoma (occasional) + Pigmented epithelioid melanocytoma + Pure mensenchymal and mixed Mullerian tumors of uterus + Tuberous sclerosis complex components + Adrenal cortical adenoma / carcinoma + Sex cord stromal tumor +

Expression of S100 in Neoplasms

Positive staining: Melanoma (95%); S100- often had prior S100+ metastases, often are ocular Sex cord stromal tumors MPNST (50%) PEComas Adenoid cystic carcinoma Chondroid tumors Chordoma Clear cell sarcoma Dendritic cell tumors Glial tumors Granular cell tumor Langerhans cell tumors Lipogenic tumors Rosai-Dorfman disease Myoepithelial tumors Neural tumors Paragangliomas: sustentacular cells Mycobacterial spindle cell pseudotumor in lymph nodes

Negative staining: Fibroblasts, fibroblastic reticulum cells, perineural cells Xanthoma Atypical fibroxanthoma Cardiac sarcomas Myofibroblastoma Solitary fibrous tumor

CAP Approved Molecular Tests  BRAF mutations: 35%, V600E (92%) and V600K (8%), which are candidates for BRAF inhibition as a therapeutic strategy.  NRAS mutations: Candidates for MEK inhibitor therapy.  Kit mutations: Candidates for tyrosine kinase kit inhibitor, imatinib mesylate.

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Head and Neck Melanoma (Excluding Ocular Melanoma): United Kingdom National Multidisciplinary Guidelines Recommendations:  At-risk individuals should be warned about the correlation between ultraviolet radiation (UVR) exposure and skin cancer, and should be given advice on UVR protection. (R)  Dermatoscopy can aid in the diagnosis of cutaneous melanoma. (R)  Histological examination after biopsy is essential to confirm the diagnosis and the tumour thickness. (G)  Excisional biopsy is method of choice. (G)  Staging investigations can be performed for both regional and distant disease. (R)  CT and/or MRI is recommended for patients with high-risk melanoma. (G)  Patients with signs or symptoms of relapse should be investigated by imaging. (R)  Imaging of the brain should be performed in patients who have stage IV disease. (G)  Patients with melanoma of unknown primary should be thoroughly examined and investigated for a potential primary source. (R)  Primary cutaneous invasive melanoma should be excised with a surgical margin of at least 1 cm. (G)  The maximum recommended excision margin is 3 cm. (R)  The actual margin of excision depends upon the depth of the melanoma and its anatomical site. (G)  US-guided FNA or core biopsy of suspected lymphadenopathy is more accurate than 'blind' biopsy. (R)  Open biopsy should only be performed if FNA or core biopsy is inadequate or equivocal. (R)  Prior to lymph node dissection, staging by CT scan should be carried out. (R)  If parotid disease is present without neck involvement, both parotidectomy and neck dissection should ideally be performed. (R)  There is no role for elective lymph node dissection. (R)  Sentinel lymph node biopsy (SLNB) can be considered in stage IB and above by specialist skin cancer multidisciplinary teams. (G)  Patients should be made aware that SLNB is a staging procedure, and should understand that it has, as yet, no proven therapeutic value. (R)  All patients with cutaneous melanoma should have their original tumour checked for BRAF gene status, and their subsequent targeted biological therapy based on this. (R)  Patients who develop brain metastases should be considered for stereotactic radio-surgery. (R) Ahmed OA1, Kelly C2. J Laryngol Otol. 2016 May;130(S2):S133-S141. 82

Head and Neck Melanoma (Excluding Ocular Melanoma): United Kingdom National Multidisciplinary Guidelines

Recommendations:  Histological examination after biopsy is essential to confirm the diagnosis and the tumor thickness. (G)  Excisional biopsy is method of choice. (G)  US-guided FNA or core biopsy of suspected lymphadenopathy is more accurate than 'blind' biopsy. (R)  Open biopsy should only be performed if FNA or core biopsy is inadequate or equivocal. (R)  Sentinel lymph node biopsy (SLNB) can be considered in stage IB and above by specialist skin cancer multidisciplinary teams. (G)  All patients with cutaneous melanoma should have their original tumor checked for BRAF gene status, and their subsequent targeted biological therapy based on this. (R)

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