医网情深：纽约病理实习有趣病例总结之“胸膜间皮瘤”——在美国行医之路系列 (刀评：这是我在医院工作中做冰冻切片的一例有趣病例，给两位实习医生从冰冻切片、细胞涂片、组织学诊断/鉴别诊断要点、免疫组化染色要点，等方面做了简要介绍。ZT回家仔细搜索研读了文献资料，写出简明扼要的文摘，体现出经过在MGH多年科研的优秀素质。） Case study: Malignant pleural mesothelioma TZ   Mesothelioma is a rare neoplasm that arises from mesothelial cells lining body cavities including the pleura, pericardium, peritoneum, and tunica vaginalis. Malignant pleural mesothelioma is the most common form of mesothelioma, and is strongly associated with asbestos exposure either occupational or residential. There are 2,000 to 3,000 new cases per year in the United States, and 28,000 to 43,000 people die from malignant pleural mesothelioma worldwide every year [1]. The overall survival time is from 4 to 27 months, depending on subtype. The median age at diagnosis is between 63 and 70 years old and 75 to 87% are male [2].     Loss of expression of BRCA1 associated protein (BAP1) or methylthioadenosine phosphorylase (MTAP) or homozygous deletion of cyclin dependent kinase inhibitor 2A (CDKN2A) (p16) by FISH helps to distinguish reactive mesothelial proliferation from malignant pleural mesothelioma [3].   Case summary A 56-year-old female presented with cough and shortness of breath. X-ray and CT scan showed right pleural effusion, focal nodular thickness of right pleura, and normal lung. CT-guided core biopsy and pleural biopsy by video assisted thoracoscopic surgery were performed. Frozen sections, smear cytology, H&E staining, and immunohistochemistry of WT1, calrectin, CK5/6, TTF-1, and CK7 were utilized to differentiate malignant pleural mesothelioma from metastatic lung adenocarcinoma. The final diagnosis is malignant pleural mesothelioma.   Pathophysiology • Hypotheses for how asbestos causes malignant pleural mesothelioma include o Toxic oxygen radical generation o Chronic pleural irritation o Persistent kinase mediated signaling • Asbestos fibers exert cytotoxic and genotoxic effects • Long and thin fibers associated with higher mutagenesis • Erionite and zeolite fibers are specifically linked to malignant pleural mesothelioma and likely more potent carcinogens than other asbestos fibers • Common inactivated tumor suppressors in malignant pleural mesothelioma: BAP1, neurofibromin 2 (NF2), CDKN2A • Malignant mesothelioma in situ has high risk of developing invasive mesothelioma   Clinical features • Shortness of breath • Chest wall pain, pleurisy • Cough • Weight loss • Recurrent unilateral pleural effusion; might be hemorrhagic • Occasionally asymptomatic when discovered at early stage   Diagnosis • Pleural thickening or recurrent pleural effusion on chest X-ray followed up with contrast enhanced chest CT scan • Thoracocentesis acquiring pleural fluid for cytology o In the past, was often considered not sufficient for definite diagnosis o With BAP1 and MTAP immunostaining and FISH for homozygous deletion of CDKN2A, diagnosis of malignant pleural mesothelioma possible on at least a subset of fluids [4] • Pleural biopsy: video assisted thoracoscopic surgery (preferred), CT guided core biopsy, open biopsy • Mediastinoscopy with lymph node sampling

Figure 1. (A) Moderate right sided pleural effusion (arrow); (B) Irregular thickening of pleura (black arrowheads) throughout right hemithorax including fissure (white arrow). Contributed by Anja C. Roden, M.D.   Laboratory • Serology for mesothelin and fibulin-3 might be useful as screening markers for malignant pleural mesothelioma. Osteopontin lacks specificity.   Gross description • Thick rind of pleura possibly extending into fissures • Studding of pleura • Multiple pleural nodules • Single pleural based mass rare (localized malignant pleural mesothelioma) • Optimal orientation of specimen important for assessment of invasion: pleural sections submitted perpendicular to surface; should contain entire pleural thickness together with adjacent structures such as lung, adipose tissue or skeletal muscle

Microscopic description • Thickened pleura due to neoplastic cells and desmoplastic stromal reaction with or without necrosis • Invasion of neoplastic cells into adipose tissue, skeletal muscle or lung • Tumefactive growth of malignant cells • Malignant mesothelial cells are either of epithelioid (epithelioid subtype) or spindled (sarcomatoid / desmoplastic subtype) cytology or a combination thereof (biphasic) • Epithelioid subtype most common, 36 - 53%; sarcomatoid subtype, 12 - 27%; biphasic 11 - 19% [5] • Epithelioid: o Cytology usually bland o Patterns: solid, acinar (glandular), tubulopapillary, trabecular, micropapillary, microcystic (adenomatoid), clear cell, deciduoid, small cell o Psammoma bodies might be present in any pattern • Sarcomatoid: o Haphazard growth of malignant cells o Desmoplastic variant: paucicellular, usually bland appearing spindle cells growing in a storiform pattern in a collagenized background with stromal invasion and possible bland necrosis

Figure 3. Frozen sections. (A) The 56-year-old female who presented with cough and shortness of breath. Papillary tumor invades into adipose tissue (x10); (B) The 56-year-old female who presented with cough and shortness of breath. Epithelioid tumor cells. Contributed by Gang He, M.D., Ph.D. (x40); (C) Full thickness of pleura with a diffuse infiltrate of atypical cells invading into adipose tissue (arrow) consistent with a malignant process. This biopsy is well oriented as it represents full thickness of pleura with pleural surface (S) and adipose tissue (A) to evaluate for invasion (x10); (D) Invasion of atypical epithelioid cells into adipose tissue (arrow) consistent with a malignant process (x40).

Cytology description • In general, only epithelioid malignant pleural mesothelioma shed into pleural effusion • Epithelioid cells in sheets, clusters, morules, papillae • Usually bland cytology, can be pleomorphic • Psammoma bodies possible

Table 1. Commonly used immunohistochemical stains for the diagnosis of malignant pleural mesothelioma [6, 7]. aLower percentages are described in sarcomatoid subtype and higher percentages in epithelioid subtype; bGATA3, strong, diffuse expression in sarcomatoid neoplasm argues for sarcomatoid malignant mesothelioma.   Negative stains • Loss of nuclear BAP1 or cytoplasmic (or substantially weaker than positive internal control) MTAP expression confirm malignancy but are not specific for malignant pleural mesothelioma • Loss of cytoplasmic MTAP expression is a reasonable alternative for homozygous deletion of CDKN2A; if MTAP expression is preserved but morphology and immunophenotype are highly suspicious for malignant pleural mesothelioma, CDKN2A by FISH might be performed [8] • Aberrant expression of CK20 rare

Figure 6. (A) The neoplastic cells of the 56-year-old female patient were focally positive for WT1 (nuclear staining; cytoplasmic staining is not consistent with mesothelioma; x40, IHC); (B) The neoplastic cells of the 56-year-old female patient were positive for calretinin (nuclear and cytoplasmic staining). Contributed by Gang He, M.D., Ph.D (x40, IHC); (C) Spindle cells express diffusely CK5 (x40, IHC); (D) The neoplastic cells are strongly and diffusely positive for CK5/6 (membranous and cytoplasmic staining; x100, IHC); (E) The neoplastic cells are strongly and diffusely positive for D2-40 (membranous stain; x40, IHC); (F) Spindle cells express strongly and diffusely keratin (x40, IHC); (G) Spindle cells lost expression of BAP1 (preserved nuclear expression of BAP1 in benign cells which is an important positive internal control; x40, IHC); (H) Spindle cells lost expression of MTAP (preserved nuclear and cytoplasmic expression of MTAP in benign cells which is an important positive internal control). Contributed by Anja C. Roden, M.D. (x40, IHC).   Molecular / cytogenetics description • In a subset of malignant pleural mesothelioma o Somatic inactivating mutations in BAP1 gene o Germline mutations in BAP1 gene o Homozygous deletion of CDKN2A o Deletion of NF2 (53 - 76%)   Differential diagnosis • Lung adenocarcinoma directly extending into pleura: o Expression of TTF1 or Napsin A in a subset o Expression of carcinoma markers (e.g., pCEA, MOC31, BerEP4)

Figure 7. (1) TTF1 (nuclear) and Napsin A (cytoplasma) are strongly positive in lung adenocarcinoma. Contributed by Andrey Bychkov, M.D., Ph.D. (IHC, x40); (2) CK7 is positive in lung adenocarcinoma. Contributed by Kruti P. Maniar, M.D. (IHC, x10).

• Metastatic disease: o Immunohistochemistry and history required o Metastatic adenocarcinoma ▪ Expression of MOC31, BerEP4, B72.3, pCEA ▪ Expression of TTF1 or Napsin A (suggest lung origin) o Metastatic squamous cell carcinoma ▪ Keratinization or intercellular bridges in a subset ▪ Expression of p40 o Metastatic sarcomatoid carcinoma ▪ Expression of claudin 4 might be helpful o Sarcoma (primary or metastatic) ▪ Lack of expression of keratin ▪ Expression of lineage specific markers ▪ Presence of disease defining molecular alterations such as t(X;18) and rearrangements of SS18 gene o Metastatic carcinoma ▪ Expression of carcinoma markers in carcinoma component ▪ Possible expression of lineage specific markers in sarcoma component o Malignant melanoma ▪ Expression of markers of melanocytic differentiation (e.g., S100, SOX10, MelanA) o Epithelioid hemangioendothelioma (EHE) ▪ Intracytoplasmic vacuoles containing red blood cells ▪ Expression of CAMTA1 ▪ Expression of vascular markers (CD31, CD34, FLI1, ERG) • Reactive mesothelial proliferation: o Lack of invasion, tumefactive or haphazard growth o Expression of BAP1 and MTAP preserved o Wild type of CDKN2A    References: 1. Fels Elliott, D.R. and K.D. Jones, Diagnosis of Mesothelioma. Surg Pathol Clin, 2020. 13(1): p. 73-89. 2. Saddoughi, S.A., Z.M. Abdelsattar, and S.H. Blackmon, National Trends in the Epidemiology of Malignant Pleural Mesothelioma: A National Cancer Data Base Study. Ann Thorac Surg, 2018. 105(2): p. 432-437. 3. Jaurand, M.C. and J. Fleury-Feith, Pathogenesis of malignant pleural mesothelioma. Respirology, 2005. 10(1): p. 2-8. 4. Kinoshita, Y., et al., A combination of MTAP and BAP1 immunohistochemistry in pleural effusion cytology for the diagnosis of mesothelioma. Cancer Cytopathol, 2018. 126(1): p. 54-63. 5. Zhuo, M., et al., Survival analysis via nomogram of surgical patients with malignant pleural mesothelioma in the Surveillance, Epidemiology, and End Results database. Thorac Cancer, 2019. 10(5): p. 1193-1202. 6. Le Stang, N., et al., Differential Diagnosis of Epithelioid Malignant Mesothelioma With Lung and Breast Pleural Metastasis: A Systematic Review Compared With a Standardized Panel of Antibodies-A New Proposal That May Influence Pathologic Practice. Arch Pathol Lab Med, 2020. 144(4): p. 446-456. 7. Klebe, S., et al., An immunohistochemical comparison of two TTF-1 monoclonal antibodies in atypical squamous lesions and sarcomatoid carcinoma of the lung, and pleural malignant mesothelioma. J Clin Pathol, 2016. 69(2): p. 136-41. 8. Kinoshita, Y., et al., A combination of MTAP and BAP1 immunohistochemistry is effective for distinguishing sarcomatoid mesothelioma from fibrous pleuritis. Lung Cancer, 2018. 125: p. 198-204.