ESSENTIALS OF

LUNG TUMOR
CYTOLOGY

Gia-Khanh Nguyen
2008

1
ESSENTIALS OF
LUNG TUMOR
CYTOLOGY

Gia-Khanh Nguyen, M.D.

Professor Emeritus
Laboratory Medicine and Pathology
University Of Alberta
Edmonton, Alberta, Canada

Copyright by Gia-Khanh Nguyen

Revised first edition, 2008
First edition, 2007. All rights reserved. This book was legally deposited at the Library
and Archives Canada. ISNB: 0-9780929-0-2

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TABLES OF CONTENTS
Table of contents 3
Preface 4
Dedication 5
Acknowledgement and Related material 6
Key to abbreviations 7
Chapter 1: Cytologic investigations of lung tumors 8
Chapter 2: Usual lung cancers 18
Chapter 3: Neuroendocrine carcinomas 38
Chapter 4: Other primary tumors and tumorlike lesions 49
Chapter 5: Metastatic cancers 65
Chapter 6: Pleural tumors 77
PREFACE

Cytology plays a very important role in the diagnosis of lung cancers. The monograph
“Essentials of Lung Tumor Cytology “ is the result of my experience gained in over 20
years of active involvement in the cytodiagnosis of lung tumors at the University of
Alberta Hospital, Edmonton, Alberta, Canada. It is written for practicing pathologists in
community hospitals, residents in pathology and cytotechnologists who are interested in
making safe and accurate cytodiagnoses of important tumors of the lung and pleura.
The text is concise and illustrations are abundant. Several of histologic images are
included for cytohistologic correlation. In the first edition of the monograph (2007),
cytodiagnostic criteria of lung tumors were presented. In this revised edition,
immunocytochemical features of lung tumor cells that are important for tumor typing
and differential diagnosis are stressed. A number of important references are listed at
the end of each chapter for further consultation.

This monograph was prepared by myself. Therefore, a few typographical errors may be
found in it. For improvement of its future editions, constructive comments and
suggestions from the reader will be highly appreciated.

Gia-Khanh Nguyen, M.D.

Surrey, BC, Canada
[email protected]
Winter 2008

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To my family with love

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ACKNOWLEDGEMENTS
I wish to thank Dr. Jason Ford and Mrs. Helen Dyck of The David Hardwick Pathology
Learning Centre of The University of British Columbia, Vancouver, Canada for their
interest and enthusiasm for publishing this monograph online. Their superb work is
highly appreciated.

I also wish to thank my family members for their moral support over the years.

Gia-Khanh Nguyen, M.D.

RELATED MATERIAL BY THE SAME AUTHOR

Essentials of Needle Aspiration Biopsy Cytology, 1991
Essentials of Exfoliative Cytology, 1992
Essentials of Cytology: An Atlas, 1993
Critical Issues in Cytopathology, 1996
Essentials of Abdominal Fine Needle Aspiration Cytology, 2007, 2008
Essentials of Head and Neck Cytology, 2009
Essentials of Fluid Cytology, 2009
Essentials of Gynecologic and Breast Cytology, 2010

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KEY TO ABBREVIATIONS

FNA: Fine needle aspiration or Fine needle aspirate

TBFNA: Transbronchial/mucosal FNA
TTFNA: Transthoracic FNA
Pap: Papanicolaou stain
HE: hematoxylin and eosin stain
ABC: Avidin-biotin complex technique

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Chapter 1

CYTOLOGIC INVESTIGATIONS OF
LUNG TUMORS
Investigation of lung diseases using cytologic materials has a long history that can be
traced back to the 19th century. It began with the identification of exfoliated bronchial
epithelial cells in sputa by Donne in 1845 and it was followed by the description of lung
cancer cells by Walshe in 1846 and by Hampeln in 1887. Pulmonary cytology had no
remarkable developments in the early years of the 20th century until the 1950s when a
large number of papers reporting on the ability to detect and type lung cancers were
published. In the 1960s the technique of TTFNA of lung cancer under chest fluoroscopic
guidance was developed and the early years of 1980s marked the development of
TBFNA via a flexible fiberoptic bronchoscope that allowed cytologic diagnoses of
submucosal lesions and enlarged peribronchial lymph nodes.

THE RESPIRATORY TRACT

The respiratory tract is divided into upper and lower parts. The upper respiratory tract
is composed of the nose and larynx, and the lower respiratory tract consists of the
trachea and lung. The tracheobronchial tree contains cartilage and submucosal mucus-
secreting glands and is lined by a pseudostratified, ciliated columnar epithelium that
contains, in addition, goblet cells, Clara cells and Kulchitsky cells (neuroendocrine cells).

Fig. 1.1. Histology of normal tracheobronchial wall showing submucosal mucus-

secreting glands. (HE, x100).

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The bronchi ultimately branch into bronchioles that do not have cartilage and
submucosal glands. The terminal bronchioles are purely conducting ducts that divide
into respiratory bronchioles which merge into alveolar ducts and alveoli. (Fig.1.1 and
Fig. 1.2).

Fig.1.2. Normal ciliated pseudotratified columnar bronchial epithelium. (HE, x 250).

The alveoli are lined by type I and II epithelial cells. (Fig.1.3). Type I cells account for
40% of the alveolar cells, covers 95% of the alveolar surface and facilitate gas
exchange. Type II cells produce surfactant and can reconstitute the alveolar surface
after injury. The lung and the inner aspect of the thoracic cavities are covered by a
layer of mesothelial cells.

Fig.1.3. Normal lung parenchyma showing alveolar spaces. (HE, x 100).

DIFFERENT TYPES OF RESPIRATORY CELL SAMPLES

The lower respiratory tract is the target of respiratory cytology that can be studied by
one or a variable combination of the following 7 types of cell sample: sputum, bronchial

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suction, bronchial wash, bronchial brush, bronchoalveolar lavage, TBFNA and TTFNA.
Tumors of the pleura can be investigated by cytologic examination of associated serous
effusions or TTFNA that will be discussed in Chapter 6.

1. Sputum. Sputum cell samples are obtained by early morning deep cough after
mouth washing. These are excellent specimens for screening of cancers arising from
the tracheobronchial tree. Usually 3 samples collected on 3 consecutive days are
required. The commonly used fixatives are 70% ethanol and Saccomanno solution
(50% ethanol and 2% polyethylene glycol or carbowax). If the patient is unable to
expectorate properly, the sputum expectoration can be induced by inhaling nebulized
water or saline. For a sputum specimen collected in 70% ethanol, the classic “pick and
smear” technique is used. Two to 4 smears are prepared, immediately fixed in 95%
ethanol and stained by the Papanicolaou technique. The rest of the specimen is fixed in
formalin and embedded in paraffin for cell block sections. Sputum collected in
Saccomanno solution is homogenized in a blender and concentrated by centrifugation.
It can also be processed using a thin layer method. The sputum processing must be
performed under a biologic safety hood to minimize the risk of infection by inhalation.
An sputum cell sample must contain alveolar macrophages and other cells derived from
the lung. (Fig.1.4).

Fig.1.4. Adequate sputum cell sample showing alveolar macrophages. (Pap, x 500).

2. Bronchial materials
Bronchial aspiration and washing. Bronchial secretions may be aspirated from the
trachea via a tracheal tube or a tracheotomy stoma. Bronchial wash is performed during
bronchoscopy by instilling vials of 5 to 10 mL of warm normal saline into a bronchus.
The fluid is then aspirated and usually 4 cytospin smears are prepared and stained by
the Papanicolaou method. A bronchial wash from a normal individual should show a few
bronchial columnar cells admixed with polymorphonuclear leukocytes and macrophages.
(Fig. 1.5). It is often contaminated with squamous cells exfoliated from the upper
respiratory tract. Bronchial washing is contraindicated in patients with respiratory failure
or uncontrolled coughing.

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Fig.1.5. Bronchial washing showing normal bronchial epithelial cells, alveolar
macrophages and metaplastic squamous cells. (Pap, x 500).

Bronchial brushing is performed during bronchoscopy. A cytobrush is used to scrape

the surface of a bronchial lesion. The entrapped cells are transferred to a frosted slide
by circular movements. Usually 2 smears are prepared and stained by the Papanicolaou
technique. It can be done 2 to 3 times to secure an adequate number of diagnostic
cells. Cytologic material obtained by bronchial brushing contains abundant bronchial
epithelial cells and a small number of neutrophils as well as a few squamous cells
exfoliated from the upper airways (Fig. 1.6 and Fig. 1.7). Bronchial brushing is
contraindicated in patients with respiratory failure and uncontrolled coughing.

Fig. 1.6. Bronchial brushing showing 2 bronchial epithelial fragments consisting of ciliated
columnar cells with terminal plates and a benign metaplastic squamous cell. (Pap, x 500).

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Fig.1.7. Bronchial brushing showing a few columnar bronchial epithelial cells and goblet
cells with intracytoplasmic mucous vacuoles. (Pap, x 500).

Bronchoalveolar lavage (BAL). A bronchoscope is wedged into position as far as it

can advance. The distal airways are flushed with several vials of warm normal saline
totaling 300 mL. The flushed samples are then aspirated. The first sample contains
mainly bronchial secretion and is discarded. Other samples are pooled together and
usually 4 cytospin smears are prepared and stained by the Papanicolaou and/or Diff-
Quik technique. BAL reflects the cellular changes within alveolar spaces. A satisfactory
BAL cell sample should contain abundant alveolar macrophages and a few lymphocytes
and polymorphonuclear leukocytes. (Fig.1.8). The number of epithelial cell (bronchial
columnar and squamous cells) should be less than 5% of all cells present in the sample.
Differential cell counts are obtained by evaluating 200 cells. In normal, nonsmoking
individuals polymorphonuclear leukocytes account for about 1% of all cells present.
Neutrophils, up to 4%, can be found in the BAL from a cigarette smoker without any
lung disease. BAL is contraindicated in patients with respiratory failure and uncontrolled
coughing.

Fig.1.8. BAL sample from a city resident showing numerous alveolar macrophages. A
few of them contain dust and carbon particles. (Pap, x 500).

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3. Transbronchial/transmucosal fine needle aspiration. By TBFNA cell samples
from a submucosal mass lesion or a paratracheal or parabronchial lesion or enlarged
lymph node can be obtained by a 22-gauge needle via the suction tube of a flexible
bronchoscope. The sample is commonly contaminated with bronchial secretions
containing exfoliated bronchial epithelial cells and submucosal glandular cells may rarely
be seen. (Fig.1.9).

Fig.1.9. Acini of a normal bronchial submucosal gland in a TBFNA. (Pap, x 500).

An adequate TBFNA cell sample from a lymph node should show abundant lymphocytes.
(Fig.1.10). TBFNA is almost free of complications. However, transient hemoptysis is
common and pneumothorax is exceedingly rare. It is contraindicated in patients with
uncontrolled coughing, respiratory failure and bleeding disorders.

Fig.1.10. Adequate TBFNA of an enlarged peribronchial lymph node showing abundant

lymphoid cells. (Pap, x 500).

4. Transthoracic fine needle aspiration. TTFNA is used for investigation of patients

with a lung mass lesion, usually peripheral, showing no diagnostic cells in sputum,
bronchial washing and brushing, BAL and TBFNA. It is contraindicated in patients with

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chronic obstructive lung disease, uncontrolled coughing, bleeding disorders, severe
pulmonary hypertension, arterio-venous malformation and suspected hydatid cyst. The
most common complication of TTFNA is pneumothorax which is minor and detectable
by chest roentgenogram in 21-34% of patients. However, only 10% of pneumothoraces
require a chest tube drainage. Transient hemoptysis occurs in 5-10% of cases. Other
complications include hemothorax, air embolism, tumor seeding along the needle tract
and rare sudden death. An adequate TTFNA cell sample from a normal lung tissue
should show alveolar macrophages, bronchial epithelial cells and sheets of
mesothelium. (Fig.1.11).

Fig.1.11. TTFNA from a normal lung showing a large fragment of mesothelium with
folding and several alveolar macrophages. (Pap, x 500).

ANCILLARY TECHNIQUES

In recent years, with the availability of numerous commercially available antibodies

cytologic typing of lung tumors, in particular metastatic cancers, has become more
feasible. An accurate cytodiagnosis of a metastatic tumor to the lung and an
identification of a primary lung cancer arising in a patient with a malignant tumor in
remission are very important for patient management. Cytochemical and
immunocytochemical studies can be done with satisfactory results on previously stained
smears without prior destaining. However, they are best performed on formalin-fixed
minute tumor tissue fragments in cell blocks prepared from materials procured by
bronchial brushing or FNA. Any grossly identified minute tissue fragments in an FNA
should be removed and fixed in formalin for histologic, cytochemical and
immunohistochemical studies. They may also be fixed in 2% glutaraldehyde for
ultrastructural evaluation. It should be born in mind that ethanol is not a suitable
fixative for electron microscopy as it destroys cellular ultrastructures.

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SENSITIVITY, SPECIFICITY AND PREDICTIVE VALUES

The sensitivity and specificity rates and predictive values of different types of
respiratory specimen in the diagnosis of lung cancer vary with the tumor location and
the type and number of specimens. In general a combination of different types of cell
sample offers higher sensitivity and specificity rates and predictive value for a positive
result than a single sample.

Sputum cytology is more efficient in detecting cancers involving large proximal bronchi.
Its sensitivity rate is low with one specimen (27% - 41%) and when 3 samples are used
it increases to 57% - 89%. If 5 samples are used a sensitivity rate as high as 96.1%
may be reached. It is more sensitive in detecting central bronchial carcinomas than
peripheral and metastatic lung cancers, with a sensitivity rate of 70% - 85% versus
50% - 60%, according to several reported series. The sensitivity rate of bronchial
washing in the diagnosis of lung cancer varies from 61% to 76%, and that of bronchial
brushing ranges from 70% to 77%. BAL has a sensitivity rate of 37.5% in detecting
lung cancer. For TTFNA of lung cancers, the sensitivity and specificity rates are 89%
and 96%, respectively. Its positive and negative predictive values are 98% and 70%,
respectively; and a false-positive and false-negative rates are 0.85% and 6%,
respectively. For TBFNA, the sensitivity rate of the procedure alone is about 52%. When
TBFNA is combined with bronchial washing and brushing and bite biopsy its sensitivity
rate increases to 72%. The specificity rate of the biopsy technique is 70% - 74% and its
positive and negative predictive values are 100% and 53% - 70%, respectively.
Regarding benign pulmonary neoplasms a sensitivity of 78% and a specificity of 100%
by TTFNA have been documented. Other benign lung tumors are rare and most cases
with cytologic evaluation are single case reports. Therefore, their sensitivity and
specificity can not be estimated meaningfully.

For tumor typing, the cytohistologic correlation rates of sputum and bronchoscopy
cytologic materials, as reported by Johnston and Bossen, were 85% for squamous cell
carcinoma, 79% for adenocarcinoma, 30% for large cell carcinoma and 93% for small
cell carcinoma of the bronchial tree. Those investigators have also reported that the
cytohistologic correlation rates of TTFNA were 80%, 96%, 42% and 95% for squamous
cell carcinoma, adenocarcinoma, large cell carcinoma and small cell carcinoma of the
lung, respectively.

BIBLIOGRAPHY

Bedrossian CWM, Rybka DL. Bronchial brushing during fiberoptic bronchoscopy for
cytodiagnosis of lung cancer: comparison with sputum and bronchial washings. Acta
cytol. 1976;20: 446.

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Caglayan B, et al. Transbronchial needle aspiration in the diagnosis of endobronchial
malignant lesions: a 3-year experience. Chest. 2005;128: 704.

Dunbar F, Leiman G. The aspiration cytology of pulmonary hamartomas. Diagn

Cytopathol. 1898; 5:174.

Erozan YS, Frost JK. Cytopathologic diagnosis of cancer in pulmonary material: a critical
histopathologic correlation. Acta Cytol. 1970;14: 560.

French CA. Respiratory tract. In Cytology. Diagnostic principles and clinical correlates.
2nd ed, 2003. Cibas ES, Ducatman BS, eds. Philadelphia, Saunders. P. 61

Garg S, et al. Comparative analysis of various cytotechnical techniques in diagnosis of

lung diseases. Diagn Cytopathol. 2007;35:26.

Johnston WW. Cytodiagnosis of lung cancer. Principles and problems. Path Res Pract.
1986;181:1.

Johnston WW, Bossen EH. Ten years of respiratory cytopathology at Duke University
Medical Center. I. The cytopathologic diagnosis of lung cancer during the years 1970-
1974, noting the the significance of specimen number and type. Acta Cytol.1981;25:
103.

Johnson WW, Bossen EH. Ten years of respiratory cytopathology at Duke University
Medical Center. II. A comparison between cytopathology and histopathology in typing
of lung cancer during the years 1970-1974. Acta Cytol. 1981;25:499.

Koss LG, et al. pulmonary cytology-a brief survey of diagnostic results from July 1st,
1952 until December 31st, 1960. Acta Cytol. 8:104.

Ng ABP, Horak GC. Factors significant in the diagnostic accuracy of lung cytology in
bronchial washings and sputum samples. I Bronchial washings. Acta Cytol. 1983;27:
391.

Ng ABP, Horak GC. Factors significant in the diagnostic accuracy of lung cytology results
in bronchial washing and sputum samples. I. Sputum samples. Acta cytol. 27: 397.

Layfield LJ, et al. Guidelines of the Papanicolaou Society of Cytopathology for the
examination of cytologic specimens obtained from the respiratory tract. Diagn
Cytopathol.1999;21:61.

Nguyen GK, et al. Transmucosal needle aspiration biopsy via the fiberoptic
bronchoscope. Value and limitations in the cytodiagnosis of tumors and tumor like-
lesions of the lung. Pathol Annu. 1992; 27 (1):105.

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Sterrett G, et al. Tumours of lung and mediastinum. In Diagnostic cytopathology, 2nd
edition, 2003. Gray and McKee GT, eds. Churchill Livingstone, p. 71.

Pilotti S, et al. Sputum cytology for the diagnosis of carcinoma of the lung. Acta Cytol.
1982;26: 649.

Pilotti S, et al. Cytologic diagnosis of pulmonary carcinoma on bronchial brushing

material. Acta Cytol. 1982;26: 655.

Powers CN. Complications of fine needle aspiration biopsy: the reality behind myths.
Cytopathology. Chicago, Am Soc Cytol. 1996, p. 69.

Raab SS, et al. Metastatic tumors in the lung: a practical approach to diagnosis. In
Practical Pulmonary Pathology, Leslie KO and Wick MR, eds, Philadelphia, Churchill
Livingtone, 2005, p 603.

Tanaka T, et al. Cytologic and histologic correlation in primary lung cancer: a study of
154 cases with respectable tumors. Acta Cytol. 1985;29:49.

Truong et al. Diagnosis and typing of lung carcinomas by cytopathologic methods: a

review of 108 cases. Acta Cytol. 1985;29:379.

Weisbrod GL.Transthoracic percutaneous lung biopsy. Radiol Clin N Am. 1990; 28:647.

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Chapter 2

USUAL LUNG CANCERS

Bronchogenic carcinoma is the commonest cause of cancer death worldwide and it is
caused by cigarette smoking in the vast majority of cases. Lung cancers in smokers
frequently contain a typical, though not specific, molecular characteristic feature in the
form of G:C > T:A mutations in the TP53 gene that are probably caused by
benzo[a]pyrene, one of the many carcinogens in tobacco smoke. Other molecular
alterations that have been found in the pathogenesis of lung cancer include K-ras
oncogen mutations, Myc oncogen overexpression, Rb mutations and Bcl-2
protooncogene expressions.
About 215,000 new cases of lung cancer are expected to be diagnosed in 2008 in
the United States. Lung cancer usually occurs between 60 and 70 years of age and has
a male predominance, but the number of affected women is increasing. Over 90% of
usual bronchogenic carcinomas may be classified into four major histologic types:
squamous cell carcinoma, adenocarcinoma, large cell carcinoma and small cell
carcinoma.
The clinical manifestations of bronchogenic cancers have some common features:
cough, dyspnea, hemoptysis, chest pain, obstructive pneumonia and pleural effusion. A
Pancoast syndrome may be present when an apex lung cancer invades the eighth
cervical and first and second thoracic nerves. A Horner syndrome is observed if an apex
lung cancer (Pancoast tumor) invades cervical sympathetic nerves. When a lung cancer
involves the mediastinum a superior vena cava syndrome may develop.
Since the therapeutic options for small cell carcinoma and other bronchogenic
carcinomas are different, a correct identification of a small cell or a nonsmall cell
carcinoma of the lung is mandatory for patient management. Recent advances in
chemotherapy of lung cancers have also required a correct diagnosis of nonsmall cell
carcinoma subtypes (squamous cell versus nonsquamous cell carcinoma) for a more
effective treatment of inoperable tumors. In general, about 30% of all bronchogenic
carcinomas are resectable when diagnosed. The prognosis of lung cancer is poor and its
5-year survival rate is about 10% in most reported series.

SQUAMOUS CELL CARCINOMA

This tumor accounts for about 30% of all primary lung cancer. It commonly arises from
a major or segmental bronchus and invades the surrounding lung parenchyma. Central
cavitation may occur. Bronchogenic squamous cell carcinoma may be well- or poorly
differentiated. (Fig. 2.1 and Fig. 2.2). A well-differentiated neoplasm shows keratin
pearls and intercellular bridges. A poorly differentiated tumor may mimic a poorly
differentiated adenocarcinoma or large cell carcinoma histologically.

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The cytologic manifestations of a well-differentiated squamous cell carcinoma in the
sputum and in materials obtained by bronchial washing, bronchial brushing and FNA are
basically similar and consist of malignant keratinizing squamous cells present
predominantly singly. The individual tumor cell shows well-defined cytoplasmic
contours, orangeophilic, eosinophilic or basophilic, densely granular cytoplasm and
hyperchromatic, “ink-dark” pleomorphic nuclei. Tumor cells forming epithelial pearls and
intercellular bridges may be seen. A poorly differentiated tumor shows cohesive clusters
on non-keratinizing malignant epithelial cells with ill-defined, opaque cytoplasm and
hyperchromatic nuclei with prominent nucleoli. (Fig. 2.3 to Fig. 2.9).

Fig. 2.1. Histology of a bronchogenic well-differentiated squamous cell carcinoma.

(HE,x 250)

Fig. 2.2. Histology of a bronchogenic poorly differentiated squamous cell carcinoma.

(HE, x 250).

Histologic subtypes of bronchogenic squamous cell carcinoma such as clear cell or small
cell variants may yield cells mimicking those of a large cell carcinoma, adenocarcinoma
of the lung with extensive clear cell change and metastatic clear cell carcinoma from the

19
kidney and ovary or cells derived from a small cell lung cancer. In these situations
immunocytochemical studies of the obtained neoplastic cells may yield important
information for a more accurate tumor typing. Most lung squamous cell carcinomas
express high molecular weigh keratin, CK5/6, p63 and carcinoembryonic antigen (CEA),
many react to low molecular weigh keratin antibody and only a few express thyroid
transcription factor-1 (TTF1) and CK7. Therefore, cells derived from a bronchogenic
squamous cell carcinoma are practically positive for CK5/6 and p63 and negative for
CK7 and TTF1; while those of a bronchogenic adenocarcinoma and large cell carcinoma
usually express CK7 and TTF1. Cells derived from a small cell lung cancer are positive
for TTF1 and neuroendocrine markers (chromogranin and synaptophysin). Renal cell
carcinoma cells stain weakly positively with CK7 and react strongly positively with
vimentin and renal cell carcinoma antibodies. Cells from an ovarian carcinoma are
positive for CA125, vimentin, estrogen receptor and negative for CEA.

Fig. 2.3. Necrotic and viable keratinized malignant squamous cells in sputum of a
patient with a well-differentiated bronchogenic squamous cell carcinoma. (Pap, x 500).

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Fig. 2.4. Sputum cell block from the same case showing single and loosely clustered
keratinized malignant squamous cells. (HE, 250).

Fig. 2.5. A syncytial cluster of malignant epithelial cells in the sputum of a patient with a
poorly differentiated bronchogenic squamous cell carcinoma. (Pap, x 500).

Fig. 2.6. Sputum cell block section from the same case (Fig. 2.5) showing fragments of
nonkeratinized malignant squamous epithelium. (HE, x 250).

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Fig. 2.7. Bronchial brushing from a bronchogenic well-differentiated squamous cell
carcinoma showing isolated keratinized malignant squamous cells. (Pap, x 500).

Fig. 2.8. TBFNA from a bronchogenic well-differentiated squamous cell carcinoma

showing dyshesive keratinized tumor cells. (Pap, x 500).

Fig. 2.9. TTFNA from a bronchogenic poorly differentiated squamous cell carcinoma
showing a cohesive cluster of nonkeratinized cancer cells. (Pap, x 500).

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ADENOCARCINOMA

Bronchogenic adenocarcinoma accounts for about 30% of all primary lung cancers.
75% of the tumors arise from the lung periphery and present radiologically as a “coin
lesion”. In the remaining 25% of the cases it is located in a lobar or segmental
bronchus. Histologically, the tumor may be well- or poorly differentiated. A well-
differentiated adenocarcinoma is characterized by monomorphic malignant glandular
cells with conspicuous nucleoli in acinar and papillary patterns. A poorly differentiated
tumor is composed of pleomorphic malignant cells with prominent nucleoli arranged in
solid pattern and focal glandular formation and mucus production are present. (Fig.
2.10 and Fig. 2.11).

Fig. 2.10. Histology of a bronchogenic well-differentiated adenocarcinoma. (HE, x 250).

Fig.2.11. Histology of a bronchogenic poorly differentiated adenocarcinoma. (HE, x

250).

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The cytologic manifestations of bronchogenic adenocarcinomas are similar in sputum
and in materials obtained by bronchial washing and brushing and FNA. The malignant
glandular cells are present predominantly in small groups with acinar arrangement or in
large clusters. Cells from a well-differentiated tumor show fairly uniform nuclei with
smooth nuclear contours and conspicuous nucleoli. Cells from a poorly differentiated
adenocarcinoma are more pleomorphic and show single or multiple macronucleoli.
Intracellular mucus may be demonstrated with mucicarmine or periodic acid-Schiff
(PAS) stain with prior diastase digestion. (Fig. 2.12 to Fig. 2.15).

Fig. 2.12. A bronchogenic well-differentiated adenocarcinoma showing in sputum

clustered monomorphic tumor cells with vacuolated cytoplasm and conspicuous
nucleoli. (Pap, x 500).

Fig. 2.13. A bronchogenic poorly differentiated adenocarcinoma showing in sputum

clustered pleomorphic malignant glandular cells with prominent nucleoli. (Pap, x 500).

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Cells from a bronchogenic adenocarcinoma contain intracytoplasmic mucin and stain
positively with PAS and with PAS with prior diastase digestion. From the
immunocytochemical point of view, these cells are CEA, CK7, villin and TTF1 positive
and CK20 negative.

Fig. 2.14. Sputum cell block showing a cluster of malignant glandular cells with
vacuolated cytoplasm. (HE, x 250).

Fig. 2.15. TTFNA from a bronchogenic adenocarcinoma showing a cohesive cluster of

malignant glandular cells with prominent nucleoli. (Pap, x 500).

Bronchioloalveolar carcinoma is a rare subtype of lung adenocarcinoma and it has

not been definitely linked to cigarette smoking. It accounts for 1-5% of primary lung
cancers and can be unifocal or multifocal. The tumor is characterized by cuboidal or low
columnar tumor cells with conspicuous nucleoli growing along preexisting alveolar walls.
It can be mucinous or nonmucinous and intranuclear cytoplasmic inclusions may be

25
present. (Fig. 2.16 and Fig. 2.17). In sputum, small cuboidal tumor cells with oval nuclei
are seen predominantly in tridimensional clusters. In materials obtained by bronchial
brushing or FNA the tumor cells are commonly seen in large monolayered sheets with
nuclear crowding and overlapping. Intranuclear cytoplasmic inclusions may be noted.
(Fig. 2.18-Fig. 2.20). Cells from a mucinous bronchioloalveolar carcinoma are CK7 and
CK20 positive and TTF1 negative. Tumor cells from a non-mucinous tumor may express
surfactant proteins (SP-A, pro-SP-B, pro-SP-C).

Fig. 2.16. Histology of a non-mucinous bronchioloalveolar carcinoma. (HE, x 250).

Fig. 2.17. Histology of a mucinous bronchioloalveolar carcinoma. (HE, x 250).

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Fig. 2.18. Nonmucinous bronchioloalveolar carcinoma showing in sputum a cohesive
cluster of tumor cells with nuclear crowding and molding. (Pap, x 500).

Fig. 2.19. Mucinous bronchioloalveolar carcinoma showing in TTFNA a cohesive sheet of

mucus-secreting tumor cells with nuclear crowding. (Pap, x 400).

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 B
Fig. 2.20. A. Bronchioloalveolar carcinoma showing in TTFNA tumor cells that are
predominantly in irregular, large, cohesive sheets. (Pap, x 100).
B. At higher magnification focal glandular spaces, crowded tumor cells with slightly
pleomorphic nuclei and conspicuous nucleoli are observed, as well as intranuclear
cytoplasmic inclusions (Pap, x 500).

SMALL CELL CARCINOMA

Small cell carcinoma or “oat cell carcinoma” accounts for about 20% of all primary lung
cancers. The tumor is related to cigarette smoking and may be associated with a
paraneoplastic syndrome (diabetes insipidus or Cushing syndrome). It arises most
commonly from major bronchi and forms a perihilar mass and has a rapid growth with
early hilar lymph node and distant metastases. About 70% of patients with small cell
carcinoma present at an advanced stage when it is detected. Rarely, a small cell
carcinoma presents as a “coin lesion”.

Histologically, the tumor has a solid growth pattern with extensive necrosis. The tumor
cells are small, two to three times the size of a mature lymphocyte and show scant
cytoplasm, oval nuclei with finely granular chromatin pattern and inconspicuous
nucleolus. Nuclear molding is a prominent feature and mitotic index is high. Tumor
necrosis is a common finding. (Fig. 2.21). In some cases the small cell lung cancer is of
intermediate cell type and it is composed of tumor cells that are larger than those of
the classic small cell carcinoma, but the tumor cells essentially show the nuclear
features of the latter. A small cell carcinoma may coexist with a nonsmall cell
carcinoma.

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Fig. 2.21. Histology of a bronchogenic small cell carcinoma (HE, x 250).

Cytologically, the tumor cells are seen singly, in groups or along mucus threads with
nuclear molding in sputum and materials obtained by bronchial washing. Most tumor
cells are necrotic and show pyknotic and darkly stained nuclei. The smear background
contains linear basophilic necrotic debris. In bronchial brushing and FNA the tumor cells
are well-preserved and display a salt and pepper chromatin pattern with inconspicuous
nucleoli. (Fig. 2.22 to Fig. 2.24).

Fig. 2.22. Lung small cell cancer showing in sputum loosely clustered small malignant
cells with scant cytoplasm, oval nuclei and no nucleoli. Focal nuclear molding is noted.
(Pap, x 500).

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Fig. 2.23. Small cell carcinoma showing in bronchial brushing tumor cells with salt and
pepper chromatin pattern and linear, basophilic nuclear debris. (Pap, x 500).

Fig. 2.24. Small cell carcinoma, intermediate cell type showing larger tumor cells and
linear basophilic nuclear debris. (Pap, x 500).

About 90% bronchogenic small cell carcinomas are chromogranin, synaptophysin,

CD56 and TTF1 positive.

LARGE CELL CARCINOMA

Large cell carcinoma constitutes about 10% of all bronchogenic carcinomas. Most of
these tumors arise from segmental or lobar bronchi. The histologic diagnosis of large
cell carcinoma is a diagnosis of exclusion: the tumor does not show any patterns
characteristic for a squamous cell carcinoma, adenocarcinoma or small cell carcinoma.
Histologically, the tumor is composed of large malignant cells with abundant, granular

30
cytoplasm and macronucleoli. By electron microscopy large cell carcinoma almost
always shows focal squamous or glandular differentiation.

In cytologic material of all types (sputum, bronchial washing and brushing, FNA) the
tumor cells are seen singly and in loose or cohesive aggregates. These are large
malignant cells with variably abundant cytoplasm, large nuclei with single or multiple
eosinophilic macronucleoli. (Fig. 2.25 and Fig. 2.26). Cells from a bronchogenic large
cell carcinoma are usually CEA, CK7 and TTF1 positive, and CK20 negative.

Fig. 2.25. Single and clustered large tumor cells with single or multiple macronucleoli in
bronchial washing of a bronchogenic large cell carcinoma. (Pap, x 500).

Fig. 2.26. A cohesive cluster of large tumor cells from a bronchogenic large cell
carcinoma in a TBFNA showing single and multiple macronucleoli. (Pap, x 500).

31
Giant cell carcinoma is a rare variant of large cell carcinoma (1%) with very poor
prognosis. Histologically, it is characterized by giant, bizarre malignant cells with single
or multiple nuclei. The tumor yields in sputum and in materials obtained by bronchial
washing and brushing or FNA single and loosely clustered giant, bizarre malignant cells
with variably abundant cytoplasm, single, multiple and lobulated nuclei with
macronucleoli. (Fig. 2.27 and Fig. 2.28).

Fig. 2.27. Histology of a bronchogenic giant cell carcinoma showing bizarre

multinucleated giant malignant cells. (HE, x 250).

Fig. 2.28. A multinucleated large malignant cell in bronchial brushing of a giant cell
carcinoma of the lung. (Pap, x 500).

DIAGNOSTIC PITFALLS

Cytologic diagnosis of lung cancers is compounded with diagnostic pitfalls. Reactive,

hyperplastic or regenerative bronchial epithelial cells, reactive alveolar lining cells,
atypical metaplastic squamous cells may be mistaken for malignant cells. Hyperplastic

32
bronchial epithelial cells in patients with chronic obstructive pulmonary disease may
form tridimensional clusters with smooth contours or Creola bodies. Patients with viral
pneumonitis may exfoliate reactive bronchial epithelial cells in tridimensional clusters
with prominent nucleoli, mimicking cells derived from a bronchogenic adenocarcinoma.
These cells usually disappear within 2 weeks after the recovery of the lung infection.
Patients receiving hyperbaric oxygen therapy for respiratory failure may exfoliate highly
atypical reactive alveolar cells mimicking malignant glandular cells. Radiation and
chemotherapy may also induced cellular changes, readily mistaken for cancer cells (Fig.
2.29-Fig. 2.37). Those above-mentioned cells lack unequivocal cytologic features of
malignant cells such as a high nuclear:cytoplasmic ratio, irregular nuclear contours and
hyperchromatic coarsely granular chromatin clumping. Vegetable cells of food origin
may sometimes be mistaken for malignant squamous cells by an inexperienced
observer. A thick cell wall of a vegetable cell is the clue for a correct cytodiagnosis.
Hyperplastic reserve cells and lymphocytes may be mistaken for cells derived from a
small cell carcinoma by an inexperienced observer.

Fig. 2.29. Reactive bronchial epithelial cells seen in bronchial brushing of a patient with
viral pneumonitis. (Pap, x 500).

33
Fig. 2.30. Reactive/regenerative bronchial epithelial cells in bronchial brushing of a
patient with viral pneumonitis. (Pap, x 500).

Fig. 2.31. Hyperplastic bronchial epithelial cells forming a Creola body. (Pap, x 500)

Fig. 2.32. A Creola body seen in the sputum of a patient with chronic bronchitis. (Pap, x
250).

34
Fig. 2.33. A cluster of hyperplastic reserve cells showing small cuboidal cells with scant
cytoplasm and focal nuclear molding. (Pap, x 500).

Fig. 2.34. A cluster of hyperplastic alveolar cells in bronchial washing of a patient

recovering from a diffuse alveolar cell damage. (Pap, x 500).

35
Fig. 2.35. Atypical metaplastic squamous cells in bronchial brushing. (Pap, x 500).

Fig. 2.36. Highly atypical or suspicious epithelial cells in sputum of a patient receiving
radiation therapy for mediastinal germ cell tumor. (Pap, x 500).

Fig. 2.37. Highly atypical epithelial cells of probable alveolar origin in sputum of a
patient receiving chemotherapy for acute myelogenous leukemia. (Pap, x 500).

BIBLIOGRAPHY

Colby TV, et al. Tumors of the lower respiratory tract. In Atlas of tumor pathology, 3rd
series, 1995. Washington DC, Armed Forces Institutes of Pathology.

Erozan YS. Cytopathology in pulmonary biopsy procedures. In Biopsy techniques in

pulmonary disorders. Wang KP, ed. New York, Raven Press, 1989, p 139.

36
Geisinger KR, et al. Localized lung diseases. In Modern cytopathology. Philadelphia,
Churchill Livingston, 2004, p 399.

Jemal A, et al. Cancer statistics, 2008. CA Cancer J Clin. 2008; 58:71-96.

Johnston WW. Cytodiagnosis of lung cancer. Principles and problems. Pathol Res Pract.
1986; 181:1.

Koss LG, Melamed MR. Tumors of the lung: conventional cytology and aspiration
biopsy. In Koss’ Diagnostic Cytology and Its Histopathologic Bases, Koss LG and
Melamed MR, eds. 5th ed, 2006. Philadelphia, Lippincott Williams & Wilkin, p 643.

Nguyen GK, et al. Transmucosal needle aspiration biopsy via the fiberoptic
bronchoscope. Value and limitations in the cytodiagnosis of tumors and tumor-like
lesions of the lung. Pathol Annu. 1992; 27(1):105.

Nguyen GK, Kline TS. Essentials of cytology. An atlas. New York, Igaku-Shoin, 1993, p
43.

Nguyen GK, et al. Cytodiagnosis of tumors of the lung by transthoracic fine needle
aspiration. Diagn Cytopathol. 2000;23:431.

Shimosato Y, Noguchi M. Pulmonary neoplasms. In Sternberg’s Diagnostic surgical

pathology, 4th ed, 2004. Mills SE, et al (eds). Philadelphia, Lippincott Williams & Wilkins.
p 1173

Singh HK, Silverman JF. Lung, chest wall and pleura. In Fine needle aspiration cytology.
Orell SR, et al, eds. 4th ed, 2005. Philadelphia, Churchill Livingston, p 227.

Tao LC. Lung, pleura and mediastinum. In Guides to Clinical aspiration biopsy, Kline TS,
ed. New York, Igaku-Shoin. 1988.

Travis WD, et al. Pathology and genetics of tumours of the lung, pleura, thymus and
heart. In WHO Classification of Tumours, Lyon, IARCPress, 2004.

37
Chapter 3

NEUROENDOCRINE CARCINOMAS
Pulmonary neuroendocrine neoplasms are one of the most complicated and confusing
topics in human pathology. The histogenesis of these neoplasms has been
controversial, and their classification has undergone several revisions. Pulmonary
neuroendocrine tumors are generally believed to arise from the epithelial
neuroendocrine cells. These neoplasms share some common features with other
neuroendocrine tumors arising from other anatomic sites, such as neuroendocrine
growth patterns (organoid, ribbon/trabecular...), positive reactions to neuroendocrine
markers or antibodies (neuron-specific enolase, chromogranin, synaptophysin, and
specific peptide hormone, such as calcitonin, serotonin, glucagon antibodies ….), and
presence of intracytoplasmic membrane-bound and dense-core neurosecretory granules
at ultrastructural levels. Several lung tumors such as small cell carcinoma, well-
differentiated adenocarcinoma of fetal type and pulmonary blastoma and a small
percentage of nonsmall cell bronchogenic carcinomas show neuroendocrine
differentiation by immunohistochemical and ultrastructural studies.

TYPICAL CARCINOID TUMOR

Typical carcinoid tumors (TCT) of the lung account for 1-2% of all primary lung cancers,
occur in all age groups (20-70 years), with a mean of 55 years, and affect men and
women equally. About 80% of TCTs are centrally located and 10-20% are found in the
periphery of the lung. Most patients with pulmonary TCTs are asymptomatic. However,
patients with tumors arising in proximal bronchi may present with dyspnea, hemoptysis
and obstructive pneumonia. 2-7% of the patients develop a carcinoid syndrome that is
due to an increased production of serotonin, and the majority of these patients have
liver metastasis. Some patients present with Cushing syndrome which is secondary to
ACTH production by the tumor. At initial diagnosis, metastasis to hilar lymph nodes is
present in about 20% of cases. TCTs usually pursue an indolent course, and the 5-year-
disease-free survival rate is about 100%.

Histology: TCT is usually covered with an intact bronchial or squamous metaplastic

epithelium and it is composed of uniform small round or cuboidal cells arranged in
neuroendocrine growth patterns. The tumor cell nuclei are oval and show a granular
chromatin pattern, conspicuous nucleoli, and a scant or moderate amount of pale, clear
or eosinophilic cytoplasm. Peripheral TCTs are well-circumscribed, non-encapsulated
and generally unrelated to the bronchial tree. These uncommon peripheral tumors
account for about 5% of all pulmonary carcinoid tumors and are usually composed of
uniformly spindle cells with oblong nuclei showing granular chromatin pattern and

38
inconspicuous nucleoli. Areas showing a TCT may be present elsewhere within the
tumor. Fewer than 2 mitoses per 2 sq. mm and no foci of necrosis are present in TCTs.
(Fig. 3.1 and Fig. 3.2).

Fig. 3.1 Histology of a typical carcinoid tumor. (HE, x 250).

Fig. 3.2. Histology of a typical carcinoid tumor. (HE, x 250).

Cytology: TCT cells may be detected in sputum and bronchial wash if the overlying
bronchial mucosa is destroyed by ulceration or tumor invasion. Bronchial brush, TTFNA
or TBFNA are effective means to diagnose carcinoid tumors. The cytologic
manifestations of a TCT in cell samples obtained by bronchial brush and FNA have
characteristic features that are diagnostic of the tumor. The tumor cells are seen singly,
in loose aggregates or syncytial clusters. They are polygonal in shape and show either a
well-defined, moderately abundant, granular cytoplasm or an ill-defined, scant, pale
cytoplasm. The nuclei are oval in shape and show a granular chromatin pattern and
conspicuous nucleoli, and nuclear molding are rarely observed. Tumor cells wrapping
around capillary blood vessels may be present. The tumor cell cytoplasm stains

39
positively with neuron-specific enolase (NSE) and chromogranin antibodies. (Fig. 3.3 to
Fig. 3.7). It is important to note that the tumor cell nuclei of central TCTs show some
similarities with those of benign bronchial glandular epithelial cells. Therefore, cautions
should be exercised when interpreting naked nuclei in cell samples taken by bronchial
brush or FNA.

A TCT may show oncocytic change and yield cells with abundant, granular and
eosinophilic cytoplasm mimicking those of a granular cell tumor. (Fig. 3.8).
Occasionally, a TCT is composed of cells with large intracytoplasmic vacuoles and it
yields in TBFNA cells mimicking those of a signet-ring cell adenocarcinoma.
Immunocytochemical staining of the tumor cells with NSE and chromogranin antibodies
will be helpful for confirmation of the neuroendocrine differentiation of the tumor.

Fig. 3.3. Typical carcinoid tumor showing in sputum monomorphic tumor cells with
round nuclei and scant cytoplasm. (Pap, x 500).

Fig. 3.4. Typical carcinoid tumor showing in bronchial brushing dyshesive monomorphic
tumor cells with plasmacytoid configuration. (Pap, x 500).

40
Fig. 3.5. Typical carcinoid tumor showing in TBFNA single and clustered monomorphic
tumor cells. (Pap, x 500).

B
Fig. 3.6. TBFNA of a typical carcinoid tumor showing tumor cells wrapping around a
capillary blood vessel. (Pap, A x 100, B x 500).

41
Fig. 3.7. Single and clustered tumor cells aspirated from a typical carcinoid tumor
showing immunopositive cytoplasmic reaction with chromogranin antibody. (ABC, x
500).

Fig. 3.8. Carcinoid tumor oncocytic change showing clustered tumor cells with
abundant, eosinophilic cytoplasm and minimally atypical nuclei. (Pap, x 500).

Peripheral TCTs with spindle cells yield in needle aspirate randomly arranged, uniform,
spindle tumor cells with oval or spindle nuclei displaying a granular chromatin pattern
and inconspicuous nucleoli. (Fig. 3.9 and Fig. 3.10). Cells from a central TCT should be
differentiated from hyperplastic reserve cells, lymphoid cells, cells from a small-cell
adenocarcinoma or small-cell carcinoma. Cells from a spindle-cell tumor may be
mistaken for those of a metastatic melanoma, spindle-cell squamous cell carcinoma,
metastatic thyroid medullary carcinoma, spindle-cell thymoma and soft tissue tumors.
Immunocytochemical staining with NSE and chromogranin and/or calcitonin and CEA
antibodies is helpful in difficult cases.

42
Fig. 3.9. Histology of a peripheral spindle cell typical carcinoid tumor. (HE, x 250).

Fig. 3.10. A peripheral carcinoid tumor showing in TTFNA dyshesive spindle tumor cells
with elongated nuclei and scant cytoplasm in no specific pattern. (Pap, x 500).

ATYPICAL CARCINOID TUMOR

Atypical carcinoid tumors (ACT) are rare neoplasms and account for less than 25% of
all pulmonary carcinoid tumors. At initial diagnosis 70% of patients with ACT already
have hilar lymph node metastasis, and distant metastasis is present in about 20% of
the cases. The treatment of choice for an ACT is surgical resection. Post-operative
adjuvant chemotherapy with or without radiotherapy has limited affects, and the 5-year
survival rate is about 70%.

Histology and Cytology. (Fig. 3.11 and Fig. 3.12). ACTs are composed of more
pleomorphic and larger tumor cells arranged in neuroendocrine patterns. Mitoses are
abundant and tumor necrosis is common. As TCT, an ACT may be covered by an intact

43
bronchial mucosa, and therefore, it may not show any tumor cells in sputum. In
materials obtained by bronchial brushing or FNA the tumor cells are seen singly and in
loose or tight aggregates. Nuclear pleomorphism with granular chromatin pattern and
conspicuous nucleoli are prominent features. 2-10 mitoses per 2 sq. mm and/or foci of
necrosis are present. An ACT may have an endobronchial component that is composed
of a TCT. In this case cell samples procured by bronchial brushing may show only cells
with features of a TCT.

Fig. 3.11. Histology of an atypical carcinoid tumor showing more pleomorphic neoplastic
cells. (HE, x 250).

Fig. 3.12. Atypical carcinoid tumor showing in TBFNA more pleomorphic tumor cells.
Small and conspicuous nucleoli are present in some tumor cells. (Pap, x 500).

In some cases, cells derived from a small-cell cancer may simulate those of a TCT.
Staining of the tumor cells with a proliferative cell marker such as Ki-67 or MIB-1 may
provide useful information for tumor grading. Over 50% of tumor cells from a lung

44
small cell carcinoma show an immuno-positive nuclear reaction while fewer than 25%
of the tumor cell nuclei derived from a TCT or ACT stain positively with this antibody.
(Fig. 3.13). For TTF1 varying results have been reported. According to some studies
cells of a TCT and ACT are usually TTF1 negative. In other studies about 30% of lung
TCTs and ACTs are TTF1 positive. Several lung carcinoid tumors are positive for CD99.

Fig. 3-13. Cell block section from TBFNA of an atypical carcinoid tumor showing positive
nuclear staining with Ki-67 antibody. (ABC, x 200).

LARGE CELL NEUROENDOCRINE CARCINOMA

Large cell neuroendocrine carcinoma (LCNC) is rare and highly aggressive tumor
occurring in adults with a median age of 64 years (range, 35 to 75 years). Most patients
are heavy smokers and ectopic hormone production is not observed. The neoplasm may
be centrally or peripherally located and averages 3 cm in greatest dimensions (range
1.3 to 10 cm). This tumor does not appear to be a specific entity and behaves similarly
to a bronchogenic large cell carcinoma.

Histology and Cytology. LCNC consists of large pleomorphic malignant cells arranged in
neuroendocrine pattern with focal rosette formation. Mitotic figures are abundant and
geographic necrosis is common. (Fig.3.14).

45
Fig. 3.14. Histology of a lung large cell neuroendocrine carcinoma. (HE, x 250).

In cell samples obtained by bronchial brushing or FNA the tumor cells are seen singly
and in loose aggregates. They are large, pleomorphic and display well-defined, granular
cytoplasm and oval nuclei with granular chromatin pattern and prominent nucleoli,
mimicking those of a large cell carcinoma. Tumor cells arranged in tridimensional
clusters may be seen. Necrotic debris, naked nuclei, nuclear streaking and tumor cells
arranged in linear pattern and in rosettes have been reported. (Fig. 3.15). Staining with
NSE and chromogranin antibodies will be helpful for confirming the neuroendocrine
differentiation of the tumor cells examined. About 50% of large cell neuroendocrine
carcinomas are TTF-1 positive.

Fig. 3.15. TTFNA of a large cell neuroendocrine carcinoma showing large, pleomorphic
malignant epithelial cells with abundant cytoplasm, oval nuclei and prominent nucleoli.
Some tumor cells show a plasmacytoid configuration. (Diff-Quik, x 400).

46
Acknowledgement: Professor N. Shapiro, Editor-in-Chief of Russian News of Clinical
Cytology journal, Moscow, Russia, has kindly granted her permission for reusing of
some parts of the text and microphotographs from the author’s paper in this book
chapter (Nguyen GK. Cytology of neuroendocrine cancer of the lung. Russian News of
Clinical Cytology. 2004; 8 (3-4):19-23).

BIBLIOGRAPHY

Anderson C, et al. Fine needle aspiration cytology of pulmonary carcinoid tumors. Acta
Cytol 190; 34:505.

Brambilla E. Classification of broncho-pulmonary cancers (WHO 1999). Rev Mal Respir.

2002; 19:409.

Colby TV, et al. Tumors of the lower respiratory tract. In Atlas of tumor pathology, 3rd
series, Washington DC, Armed Forces Institute of Pathology, 1995, p 235, 287.

Ionescu DN, et al. Nonsmall cell lung carcinoma with neuroendocrine differentiation-an
entity of no clinical or prognostic significance. Am J Surg Pathol. 2007; 31:26.

Kakinuma H, et al. Diagnostic findings of bronchial brush cytology for pulmonary large
cell neuroendocrine carcinomas. Comparison with poorly differentiated
adenocarcinomas, squamous cell carcinomas, and small cell carcinomas. Cancer (Cancer
Cytopathol) 2003; 99:247.

Lin O, et al. Immunohistochemical staining of cytologic smears with MIB-1 helps

distinguish low-grade from high-grade neuroendocrine neoplasms. Am J Clin Pathol
2003; 120:209-216.

Mitchell MI, Parker FP. Capillaries: a cytologic feature of pulmonary carcinoid tumors.
Acta Cytol 1991; 35: 183.

Nguyen GK, et al. Transmucosal needle aspiration biopsy via the fiberoptic
bronchoscope. Value and limitations in the cytodiagnosis of tumors and tumor-like
lesions of the lung. Pathol Annu 1992; 27 (1):105.

Nguyen GK. Cytopathology of pulmonary carcinoid tumors in sputum and bronchial

brushing. Acta Cytol 1995; 39:1152.

Nguyen GK, et al. Cytodiagnosis of bronchogenic carcinoma and neuroendocrine tumor

of the lung by transthoracic fine-needle aspiration. Diagn Cytopathol 2000; 23:431.

47
Ogino S, et al. Cytopathology of oncocytic carcinoid tumor of the lung mimicking
granular cell tumor. A case report. Acta Cytol 2000; 44:247-250.

Szyfelbein WM, Ross SS. Carcinoids, atypical carcinoids and small cell carcinomas of the
lung. Diagn Cytopathol 1988; 4:1.

Travis WD, et al. Pathology and genetics of tumours of the lung, pleura, thymus and
heart. In WHO Classification of Tumours, Lyon, IARCPress, 2004.

Wiatrowska BA, et al. Large cell neuroendocrine carcinoma of the lung: proposed
criteria for cytologic diagnosis. Diagn Cytopathol 2001; 24:58.

48
Chapter 4

OTHER PRIMARY TUMORS AND

TUMORLIKE LESIONS
MALIGNANT TUMORS
BRONCHIAL GLAND CARCINOMA
Bronchial gland carcinomas are rare neoplasms occuring in adult patients. These
neoplasms may manifest with hemoptysis or bronchial obstruction with distal lung
infection. The tumors account for about 1% of all primary lung cancers and consist of
two lesions: Adenoid cystic carcinoma and mucoepidermoid carcinoma.

Adenoid Cystic Carcinoma is the most common salivary gland-like tumor of the
lower respiratory tract. It accounts for about 0.2% of all primary lung cancers. The
neoplasm usually arises from the trachea, main stem bronchi or lobar bronchi. The
patient’s age ranges from 18 to 79 years. It is a less aggressive neoplasm and has a
distinct histologic pattern of growth consisting of cribiforms and glandular arrays or
tubules surrounding central spaces filled with epithelial mucin and solid foci (Fig. 4.1).
The tumor yields in bronchial brushing and TBFNA single and clustered small, round
cells with scant cytoplasm and round basophilic bodies. Tumor cells wrapping around
basophilic bodies are a diagnostic feature of the lesion. (Fig. 4.2 and Fig. 4.3).

Fig. 4.1. Histology of a bronchial adenoid cystic carcinoma. (HE, x 250).

49
 A

B
Fig. 4.2. TBFNA of a bronchial adenoid cystic carcinoma showing in A single and
clustered small cuboidal neoplastic cells and in B an eosinophilic amorphous round body
wrapped by small tumor cells. (HE, x 400).

Fig. 4.3. TTFNA from a pulmonary adenoid cystic carcinoma reveals small tumor cells
wrapping around ball-like, basophilic, amorphous bodies and a basophilic round body at
the left lower corner of the figure. (Pap, x 250).

50
Mucoepidermoid Carcinoma is a rare tumor comprising 0.1 to 0.2% of all primary
lung carcinomas. The patients range in age from 4 to 78 years but about 50% are
younger than 30 years. The tumor most commonly arises from the main or lobar
bronchus and can measures up to 6 cm in greatest dimension. Histologically, it consists
of a variable population of mucus-secreting cells, squamous cells and intermediate cells
that display no particular differentiating characteristics. Bronchial mucoepidermoid
carcinomas can be classified as low- and high-grade tumors, depending on the degree
of cellular atypia. About 75% to 80% of mucoepidermoid carcinomas arising from the
lung are of low histologic grade. A low-grade tumor yields in FNA single and clustered
benign-appearing squamous cells admixed with benign-appearing mucus-secreting cells.
A variable number of intermediate cells may be present. A high-grade tumor yields
loosely clustered malignant squamoid cells containing intracytoplasmic mucus. (Fig. 4.4
to Fig. 4.7).

Fig. 4.4. Histology of a bronchial low-grade mucoepidermoid carcinoma showing

polygonal squamoid cells admixed with glandular cells with clear cytoplasm. (HE, x
250).

51
Fig. 4.5. A low-grade mucoepidermoid carcinoma showing in FNA loosely clustered
squamoid tumor cells with some cells showing vacuolated “clear” cytoplasm. Small
cuboidal tumor cells in the lower part of the figure are of intermediate type. (Pap, x
500).

Fig. 4.6. Histology of a high-grade mucoepidermoid carcinoma of the bronchus showing

nests of invasive malignant squamous cells with pleomorphic, hyperchromatic nuclei.
Some tumor cells have a “clear” cytoplasm. (HE, x 250).

Fig. 4.7. TBFNA of a bronchial high-grade mucoepidermoid carcinoma showing

clustered malignant squamoid cells with intracytoplasmic mucus that stains positively
with PASD. (PASD, x 400).

WELL DIFFERENTIATED FETAL ADENOCARCINOMA

This rare cancer is related to cigarette smoking and commonly occurs in 5th or 6th
decade of life. It usually pursues a less aggressive clinical course. Histologically, the
tumor is composed of low-grade malignant glandular cells arranged in acinar pattern.
Focal tumor cell morulae containing intracytoplasmic neurosecretory granules, as
demonstrated by electron microscopy and by immunohistochemical staining with

52
neuron-specific enolase and chromogranin antibodies, are present. (Fig. 4.8). Only a
few tumors of this type with cytologic evaluation have been encountered in the
literature. In one case the TTFNA revealed large monolayered and folded sheets of low-
grade malignant columnar epithelial cells with clear or granular cytoplasm and uniformly
oval, small nuclei with inconspicuous nucleoli. Focal glandular arrangement may be
visualized within a tumor cell sheet. (Fig. 4.9)

Fig. 4.8. Histology of a pulmonary well-differentiated adenocarcinoma, fetal type

(WDAFT) showing an intraglandular morule of tumor cells. (HE, x 250).

Fig. 4.9. TTFNA of a lung WDAFT showing a large sheet of tumor cells with honeycomb
pattern. A round glandular space is noted. (Pap, x 250).

PULMONARY BLASTOMA
This rare lung cancer is seen in adult patients and it is related to cigarette smoking. It is
composed of fetal-type glandular elements admixed with spindle-shaped cells and
cartilage and bone may be present. A few examples of this neoplasm with cytologic

53
evaluation by TTFNA have been reported and both types of above-mentioned cells were
observed.

LUNG SARCOMA
Primary Soft-Tissue Sarcomas. Primary lung sarcomas are exceedingly rare
neoplasms and account for less than 1% of all primary lung cancers. Almost all
histologic types of soft-tissue sarcomas have been reported in the lung. Practically, a
diagnosis of primary lung sarcoma can only be made if the patient has no history of a
treated soft-tissue sarcoma, and no sarcoma is detected by extensive clinical and
diagnostic imaging studies. In one large surgical series there were one sarcoma for
every 500 bronchogenic carcinomas. Of the primary lung sarcomas, leiomyosarcoma
is the most common one, and about 100 cases of this neoplasm have been reported in
the literature. The tumor occurs mainly in adults and rarely in children. It may arise
from a bronchus or from intraparenchymal blood vessels. The cytologic manifestations
of a primary lung leiomyosarcoma in bronchial brushing and in FNA are similar and
consist of scattered loosely aggregated, elongated slightly pleomorphic, hyperchromatic,
naked tumor cell nuclei with blunt ends. Bundle of smooth muscle cells may be present
in materials obtained by bronchial brushing. (Fig. 4.10 and Fig. 4.11). Cells derived
from a fibrosarcoma or neurogenic sarcoma are morphologically similar to those of a
leiomyosarcoma. A positive cytoplasmic reaction with smooth muscle and desmin
antibodies will confirm the diagnosis of a leiomyosarcoma.

Fig. 4.10. TTFNA of a well-differentiated leiomyosarcoma of the lung showing single

and loosely clustered tumor cells with elongated nuclei with blunt ends. (Pap, x 400).

54
Fig. 4.11. Bronchial brushing from a low-grade leiomyosarcoma arising from a lobar
bronchus reveals bundles of malignant smooth muscle cells with enlarged, elongated,
oval or polygonal nuclei. (Pap, x 400).

Embryonal or alveolar rhadomyosarcoma of the lung yields malignant small round

cells with scant cytoplasm. (Fig. 4.12). A positive reaction of the tumor cell cytoplasm
with MyoD1 or myogenin antibodies will be helpful for a more accurate tumor typing.

Fig. 4.12. Primary lung embryonal rhabdomyosarcoma showing in TTFNA small round
malignant cells with hyperchromatic nuclei and scant cytoplasm. (Pap, x 400).

Pulmonary artery angiosarcoma is a rare neoplasm of adults. In one reported case

the tumor showed in bronchial washing single and clustered small malignant cells with
scant cytoplasm and hyperchromatic oval nuclei. Focal luminal formation was noted in
some tumor cell clusters. A positive reaction of the tumor cell cytoplasm with factor VIII
related antigen antibody will confirm the diagnosis of this lung cancer.

55
BENIGN LUNG TUMORS AND TUMORLIKE LESIONS
HAMARTOMA. This benign tumor most commonly occurs in 6th decade of life. It is
usually asymptomatic and often discovered incidentally by chest roentgenograms. It is
usually located in the peripheral zone of the lung. If located in a bronchus it may cause
bronchial obstruction with distal bronchial infection. The lesion is well-circumscribed,
lobulated and usually measures 2 cm in greatest dimension. It is formed by elements
that are normally present in the lung such as cartilage, fibromyxoid connective tissue,
fat, smooth muscles and respiratory epithelium. It shows in TTFNA an admixture of the
above-mentioned cytologic elements. (Fig. 4.13 and Fig. 4.14).

Fig. 4.13. Histology of a lung hamartoma. (HE, x 100).

56
 B
Fig. 4.14.TTFNA of a lung hamartoma showing in A myxoid material, chondrocytes and
clusters of benign bronchial glandular cells, and in B a large fragment of benign
cartilaginous tissue. (Pap, x 400).

GRANULAR CELL TUMOR of the lung is a rare benign neoplasm arising from the
Schwann cell. In over 90% of cases, the tumor has an endobronchial component, and
in less than 10% of patients it presents as a parenchymal lesion and appears on chest
roentgenograms as a “coin lesion”. It yields in bronchial brushing or submucosal needle
aspirate sheets of benign tumor cells with eosinophilic, granular and PAS-positive
cytoplasm and small, oval nuclei with conspicuous nucleoli. (Fig. 4.15 and Fig. 4.16).

Fig. 4.15. Histology of a bronchial granular cell tumor showing neoplastic cells with
granular and PAS-positive cytoplasm. (PAS, x 200).

57
Fig. 4.16. A thick fragment of tumor tissue in a submucosal FNA showing benign
neoplastic cells with oval nuclei and ill-defined, granular cytoplasm. (Pap, x 400).

CLEAR CELL “SUGAR” TUMOR is a rare benign lung tumor of unknown histogenesis.
It consists of spindle-shaped cells with “glycogen-rich”, clear cytoplasm. In one reported
case the tumor yielded in TTFNA large clusters of spindle cells with clear cytoplasm and
oval or elongated bland nuclei. Intracytoplasmic glycogen can be demonstrated by
staining of the tumor cells with PAS reagent. (Fig. 4.17 and Fig. 4.18).

Fig. 4.17. Histology of a benign clear cell “sugar” lung tumor showing spindle tumor
cells with clear cytoplasm and round or elongated nuclei. (HE, x 250).

58
 A

B
Fig. 4.18. TTFNA from a benign clear cell tumor of the lung showing in A a large cluster
of spindle tumor cells with oval or elongated, bland nuclei, and in B, an aggregate of
benign epithelial-like cells with ill-defined cytoplasm, round or oval nuclei and thin,
semitransparent, “clear” cytoplasm. (HE, A x 250, B x 400).

SQUAMOUS CELL AND GLANDULAR CELL PAPILLOMAS are very rare benign
endobronchial lesions that may cause hemoptysis or bronchial obstruction with distal
bronchial and pulmonary infection. Biopsy of the lesion may cause severe hemorrhage.
The squamous cell papilloma may be solitary, multiple, exophytic or endophytic. Solitary
squamous cell papilloma is seen mainly in men in their fifth decade of life and is more
commonly exophytic. It may be associated with human papilloma virus (HPV) subtypes
6 and 11, suggesting a possible pathogenetic role for the virus. HPV subtypes 16, 18
and 31/33/35 in squamous cell papillomas associated with carcinomas and in squamous
cell carcinomas have been reported, suggesting that HPV infection might be related to
tumoral progression. Depending on the histologic type, benign squamous cells and
glandular cells are seen in materials obtained by bronchial washing or brushing. The
squamous cell tumor associated with HPV infection may show histologic features of a

59
papillary condyloma and yields in bronchial cytologic materials dyskaryotic squamous
cells with perinuclear halos. (Fig. 4.19 and Fig. 4.20). The glandular cell papilloma
exfoliates benign bronchial glandular cells and cannot be identified cytologically.

B
Fig. 4.19. Histology of a solitary bronchial squamous cell papilloma associated with HPV
6 infection showing its squamous epithelial lining displaying mild dysplasia and
dyskaryotic koilocytes. (HE, A x 4, B x 250).

60
Fig. 4.20. Dyskaryotic squamous cells with one showing koilocytic change in bronchial
washing of a patient with bronchial squamous cell papilloma. (Pap, x 500).

PULMONARY AMYLOIDOSIS most commonly occurs in patients over 60 years of

age. It usually diffusely involves the submucosa of the tracheobronchial tree but it may
appear as a parenchymal mass lesion. The bronchial lesion may mimic a submucosal
tumor and it yields in bronchial brushing or TBFNA irregular masses of amorphous,
granular, waxy material that stains slightly eosinophilic or basophilic with the
Papanicolaou stain and orangeophilic with Congo red. (Fig. 4.21 and Fig. 4.22).

Fig.4.21. Histology of a bronchial amyloid deposit covered with a benign metaplastic

squamous epithelium. (HE, x 250).

61
Fig.4.22. Bronchial brushing showing irregular, ill-defined masses of amorphous, waxy,
granular and orangeophilic amyloid material. (Pap, x 500).

WEGENER GRANULOMATOSIS is a systemic necrotizing vasculitis of unknown

etiology. It is characterized by granulomatous lesions in the nose, nasal sinuses, lung
and kidney. Over 90% of patients have ANCA in their blood. Of those, 75% are C-
ANCA. Persistent bilateral pneumonitis and chronic sinusitis are prominent clinical
manifestations, and hematuria and proteinuria are indicative of renal involvement. In
the lung the granulomata may measure up to 5 cm in greatest dimension and may
mimic a neoplasm radiologically. Untreated disease is fatal, and immunosuppressive
treatment with cyclophosphamide usually results in marked improvement. A TTFNA or
bronchial brushing of the lung lesion reveals granular debris of necrotic collagen
admixed with chronic inflammatory cells. Multinucleated giant cells and epithelioid cells
may be seen.

INFLAMMATORY PSEUDOTUMOR also known as inflammatory fibroblastic

tumor of the lung is a rare lesion that usually develops after a nonspecific pulmonary
inflammation. It occurs in men or women, usually before the age of 40. Most of these
lesions are contained within the lung and appear as a circumscribed, nodular mass
consisting of an admixture of fibroblastic cells, myoepithelial cells and chronic
inflammatory cells such as lymphocytes, plasma cells and macrophages. The above-
mentioned cellular elements may be seen in TTFNA. (Fig. 4.23). The majority of these
lesions are benign, but about 5% of them are aggressive and invade adjacent
structures such as esophagus, mediastinum, diaphragm and chest walls.

62
 A

B
Fig. 4.23. TTFNA of an pseudo-inflammatory tumor of the lung reveals irregular bundles
of fibroblastic cells admixed with scattered chronic inflammatory cells. (Pap, A x 100, B
x 400).

BIBLIOGRAPHY

Awasthi A, et al. Pitfalls in the diagnosis of Wegener’s granulomatosis on fine needle

aspiration cytology. Cytopathology. 2007; 18: 8.

Colby TV, et al. Tumors of the lower respiratory Tract. In Atlas of tumor pathology, 3rd
series, 1995.

Geisinger KR, et al. Localized lung diseases. In Modern cytopathology, Geisinger KR, et
al, eds. Philadelphia, Churchill Livingstone. 2004, p 399.

63
Gray JA, Nguyen GK. Primary pulmonary rhabdomyosarcoma diagnosed by fine needle
aspiration cytology. Diagn Cytopathol. 2003; 29:181.

Husain M, Nguyen GK. Cytopathology of granular cell tumor of the lung. Diagn
Cytopathol. 2000; 23: 294.

Machicao CN, et al. Transthoracic needle aspiration biopsy of inflammatory

pseudotumors of the lung,. Diagn Cytopathol. 1989; 5:400.

Naryshskin S, Young NA. Respiratory cytology: a review of non-neoplastic mimics of

malignancy. Diagn Cytopathol. 1993; 9:89.

Micheal CW, Flint A. The cytologic features of Wegener’s granulomatosis. Am J Clin

Pathol. 1998; 110:10.

Nguyen GK. Exfoliative cytology of angiosarcoma of the pulmonary artery. Acta Cytol.
1985; 29:627.

Nguyen GK. Cytology of bronchial gland carcinoma. Acta Cytol. 1988; 32: 235.

Nguyen GK. Aspiration biopsy cytology of benign clear-cell “sugar” tumor of the lung.
Acta Cytol. 1989; 33:511.

Nguyen GK. Fine needle aspiration cytology of well-differentiated fetal adenocarcinoma

(endodermal tumor) of the lung. Acta Cytol. 2001; 45: 475.

Odashiro AN, et al. Primary lung leiomyosarcoma detected by bronchoscopy cytology.

Diagn Cytopathol. 2005; 33: 220.

Shimosato Y, Noguchi M. Pulmonary neoplasms. In Sternberg’s Diagnostic surgical

pathology. 4th ed, 2004. Mills SE, et al, eds. Philadelphia, Lippincott Williams & Wilkins,
1173.

Tao LC. Lung, pleura and mediastinum. In Guides to Clinical aspiration biopsy, Kline TS,
ed. New York, Igaku-Shoin, 1988.

Travis WD, et al. Pathology and genetics of tumours of the lung, pleura, thymus and
heart. In WHO Classification of Tumours. Lyon, IARCPress, 2004.

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Chapter 5

METASTATIC CANCERS
The lung is one of the most common sites of metastasis from extrathoracic cancers.
From 20 to 60% of individuals with extrathoracic solid cancers show, at autopsy, lung
metastases; and lung is the only site of metastasis in 15 to 25% of these cases.
Carcinomas arising from the breast, prostate, testicles and kidney, cutaneous
melanoma, Ewing sarcoma, osteogenic sarcoma and rhabdomyosarcoma frequently
metastasize to the lung.

MACROSCOPIC PATTERNS OF METASTATIC CANCERS

Metastatic cancers in the lung display some distinctive patterns of metastasis such as
multiple tumor nodules, lymphangitic, endobronchial, endovascular, solitary and pleural.
An awareness of these macroscopic patterns of metastasis is helpful for a more
accurate cytodiagnosis of secondary lung cancers. A summary of patterns of metastasis
in the lung, as described by Colby et al is summarized below.

Multiple and bilateral masses of metastatic tumor of different sizes is the most
common pattern of lung metastasis. Concomitant lymphangitic, endobronchial and
endovascular tumor deposits may also be present. This pattern of metastasis is most
commonly seen in patients with sarcoma, renal cell carcinoma, cutaneous melanoma
and colorectal carcinoma.

Lymphangitic pattern accounts for 6-8% of all lung metastases. It is characterized

by a diffuse, linear and nodular thickening of bronchovascular bundles, interlobular
septae and subpleural spaces. About 80% of the metastatic tumors are
adenocarcinomas arising from the lung, breast, gastrointestinal tract and pancreas. This
pattern of metastasis can be diagnosed by CT scan or chest roentgenograms.

Endobronchial metastasis is found at autopsy in 18-51% of patients with

extrathoracic cancers. In most cases the bronchus is invaded by metastatic cancer
deposits in adjacent lung parenchyma or lymph nodes. Metastasis involving only a
bronchus is uncommon and is found in less than 5% of patients with solid cancers
arising from the head and neck, breast, colon, kidney and soft tissue. Endobronchial
metastases may mimic a bronchogenic cancer at bronchoscopy.

Metastatic tumor embolization is not uncommon. It is found at autopsy in 2-26%

of patients with solid cancer, and it can be diagnosed during life by cytologic
examination of blood obtained by wedge pulmonary artery catheterization. Renal cell,

65
hepatocellular and gastric carcinomas, choriocarcinoma and chondrosarcoma more
commonly show this pattern of metastasis.

Solitary metastasis is not uncommon. About 1-5% of lung metastases are solitary
and 3-9% of all solitary lung nodules are metastatic deposits. Cutaneous melanoma,
renal cell carcinoma, and colonic carcinoma, breast carcinoma, urinary bladder
carcinoma, soft tissue sarcomas and non-seminomatous testicular cancers most
frequently cause of solitary lung metastasis.

Pleural metastasis is seen in the setting of lymphatic or vascular spread with

contiguous extension from parenchymal tumor deposits. It is commonly associated with
malignant pleural effusions. Macroscopically, the pleura shows multiple and scattered
tumor nodules of different sizes ranging from miliary to dominant masses. A diffuse
infiltrating pattern, as seen in pleural mesothelioma, may be observed.

CYTOLOGY OF METASTATIC CANCERS

Endobronchial metastatic cancers may exfoliate their cells in sputum and different
bronchoscopy cytologic specimens. Lung parenchymal deposits are best diagnosed with
TTFNA. BAL may show cancer cells with alveolar spread. The cytologic manifestations of
metastatic cancers to the lung are somewhat similar in different types of pulmonary cell
samples and display similar features with those of their primary cancers arising from
different anatomic locations. Clinical history and a comparison of the metastatic cancer
cells with the histologic sections or cytologic samples of the primary cancers, if
available, are of diagnostic help in the majority of cases. Diagnosis of solitary
metastasis is important for patient care, as a second or a third primary cancer may
develop in patients who had a surgically removed primary cancer many years prior. As
in the liver, diagnosis of metastatic adenocarcinoma to the lung is challenging. An
awareness of the incidences of metastasis of cancers arising from different organs or
anatomic sites can be of diagnostic help. Immunocytochemical studies of the cell
samples or aspirated minute tumor tissue fragments with selected antibodies may be
required for a more accurate tumor typing in some cases. On rare occasions, electron
microscopic studies of aspirated tumor tissue fragments are needed for differential
diagnoses. Since there are numerous histologic types of solid tumors, therefore, it
would be more convenient to discuss the cytologic manifestations of metastases from
primary cancers arising in different anatomic locations or sites.

Breast
Mammary carcinomas frequently metastasize to the lung. The tumors yield malignant
glandular cells with nonspecific features. Tumor cells arranged in linear pattern may be
observed (Fig. 5.1). These cells usually express estrogen and progesterone receptors
and CK7 and stain negatively with CK20 and TTF1 antibodies.

66
 A

B
Fig. 5.1. Metastatic mammary duct carcinoma showing in:
A. TTFNA clustered malignant glandular cells with focal nuclear crowding. Tumor cells in
linear arrangement are noted elsewhere. (Diff-Quik, x 400).
B. Tumor cells in a cell block section showing positive nuclear staining with ER antibody.
(ABC, x 200).

Thyroid
About 15% of thyroid carcinomas metastasize to the lung. Metastatic cancers are most
frequently derived from an anaplastic carcinoma then from a poorly differentiated or
well-differentiated carcinoma. Metastatic papillary carcinoma shows papillary tissue
fragments, sheets or groups of tumor cells with nuclear crowding, intranuclear
cytoplasmic inclusions and nuclear grooves. (Fig. 5.2). A follicular carcinoma yields cells
in clusters with focal acinar arrangement. A Hurthle cell carcinoma shows tumor cells
with granular cytoplasm singly and in monolayered sheets. A medullary carcinoma may
show single and clustered polygonal and/or spindle tumor cells with elongated nuclei.
Intranuclear cytoplasmic inclusions may be noted and cytoplasmic azurophil granules

67
may be observed in tumor cells stained with MGG or Diff-Quik technique. (Fig. 5.3).
Cells derived from an anaplastic carcinoma are either large pleomorphic or spindle.
Tumor cells from a papillary, follicular, Hurthle cell or insular carcinoma express
thyroglobulin and TTF1 while those of a medullary carcinoma stain positively with
calcitonin and carcinoembryonic antigen antibodies. Cells derived from an anaplastic
carcinoma may not show any positive staining reactions with thyroglobulin or TTF1
antibodies.

Fig. 5.2. Metastatic papillary carcinoma of the thyroid, follicular variant showing clustered
tumor cells displaying nuclear crowding. Intranuclear cytoplasmic inclusions are observed
in some tumor cells. (Diff-Quik, x 500).

Fig. 5.3. Metastatic thyroid medullary carcinoma to the lung showing tumor cells with
plasmacytoid configuration. (Diff-Quik, x 500).

Gastrointestinal tract, pancreas and biliary tree

Metastatic tumors from a poorly differentiated adenocarcinomas arising from the
stomach, small and large bowels, pancreas or biliary tree yields malignant cells with no

68
specific features. Staining of the tumor cells with CDX2, CK7, CK20, MUC-2 and MUC-5
antibodies may be useful for determining the site of the primary tumor. Cells from a
biliary or pancreatic tumor are usually monoclonal CEA positive, CDX2 negative, CK7
positive, CK20 negative and MUC-5 positive while those of colorectal origin are usually
CDX2 positive, CK7 negative, CK20 positive and MUC-2 positive. Cells with signet-ring
configurations are most commonly derived from a signet-ring cell carcinoma of the
stomach. Cells from a well- or moderately differentiated colonic adenocarcinoma are seen
in sheets with elongated nuclei in picket fence pattern. A large amount of necrotic debris
is commonly noted in a FNA from a metastatic colonic adenocarcinoma. (Fig. 5.4 and Fig.
5.5).

Fig.5.4. Irregular sheets of a metastatic colonic adenocarcinoma to the lung showing a

large amount of necrotic debris and irregular sheets of tumor cells with cells at
periphery arranged in palisade. (Pap, x 250).

Fig.5.5. An endobronchial metastatic colonic adenocarcinoma yields in bronchial

brushing tumor cells with nuclei at periphery arranged in palisade. (Pap, x 500).

69
Liver
Hepatocellular carcinomas commonly spread to the lung. Single and clustered polygonal
cells with granular or vacuolated cytoplasm are seen, and intracytoplasmic globular
inclusions may be observed (Fig. 5.6 and Fig. 5.7). Cells derived from a hepatocellular
carcinoma do not express CK7 or CK20. A positive staining of the tumor cell cytoplasm
with alpha-fetoprotein or HepPar 1 antibody will confirm the diagnosis of a metastatic
hepatocellular carcinoma.

Fig.5.6. Metastatic hepatocellular carcinoma showing in BAL a tumor cell with intra
cytoplasmic globular inclusion. (Pap, x 500).

Fig.5.7. Histology of moderately differentiated hepatocellular carcinoma metastatic to

the lung showing tumor cells in Fig. 5.6. Note the presence of numerous
intracytoplasmic globular inclusions. (HE, x 250).

Salivary glands
Of the salivary gland carcinomas, adenoid cystic carcinoma most commonly
metastasizes to the lung. It is characterized in FNA small hyperchromatic cells in acinar

70
arrangement. Globular bodies of amorphous, basophilic material may be present in
smear background and globular bodies wrapped with tumor cells are commonly seen.

Urinary tract and adrenal

Renal cell carcinomas (RCC) commonly metastasize to the lung. A conventional RCC
yields cells with clear or granular cytoplasm singly, in clusters and in monolayered
sheets. (Fig. 5.8). A metastatic papillary RCC yields in FNA monolayered sheets of
monomorphic tumor cells with clear or granular cytoplasm, and papillary tumor tissue
fragments with fibrovascular core may be present. RCC cells stain positively with RCC
and negatively with CK7 and CK20 antibodies.

B
Fig.5.8. Metastatic grade 3/3 renal cell carcinoma to the lung showing in TTFNA:
A. Single tumor cells with granular, thick cytoplasm and single or multiple nuclei.
B. Sheets of tumor cells with clear or granular cytoplasm and prominent nucleoli.
(A. HE, x 200; B. Pap, x 500).

Cells from a conventional RCC and an adrenal cortical carcinoma are morphologically
similar and express both cytokeratin and vimentin. Adrenal cortical carcinoma cells

71
express, in addition, melan A or A103. By electron microscopy, adrenal cortical tumor
cells show abundant intracytoplasmic smooth endoplasmic reticula (ultrastructural
features of steroid secreting cells) while those of a RCC contains intracytoplasmic fat
droplets, glycogen granules and a normal number of smooth endoplasmic reticula. A
metastatic chromophobe RCC yields cells similar to those of a conventional RCC.
Perinuclear clear spaces and positive cytoplasmic staining with colloidal iron are other
characteristic cellular features of the tumor. Abundant intracytoplasmic microvesicles
are seen by electron microscopic study of aspirated minute tumor tissue fragments.

A metastatic high-grade transitional cell carcinoma of the renal pelvis or urinary bladder
is characterized by pleomorphic malignant epithelial cells singly and in clusters. Tumor
cells with cytoplasmic extension or “tail” (cercariform cells) are commonly seen and
constitute a fairly reliable feature for this type of neoplasm. (Fig. 5.9). Urothelial cancer
cells may express uroplakin III (URO III), CK7 and CK20.

72
Fig. 5.9. A. TTFNA of a metastatic grade 3/3 transitional cell carcinoma of the urinary
bladder to the lung showing pleomorphic malignant cells (A). A few tumor cells with
cytoplasmic tails or “cercariform cells” are noted in B. (Pap, A x 500, B x 200).

Prostate
A metastatic prostatic adenocarcinoma shows clusters and sheets of small glanfular
cells with clear cytoplasm and round nuclei with prominent nucleoli. The tumor cell
cytoplasm characteristically stains positively with prostatic specific antigen antibody.
(Fig.5.10).

B
Fig. 5.10. A showing cohesive small malignant cells in bronchial brushing of an
endobronchial metastatic prostatic adenocarcinoma. The tumor cells in B stain positively
with prostatic specific antigen antibody. (A: Pap, x 500; B: ABC, x 500).

Uterus
Cervical squamous cell carcinomas frequently spread to the lung while endocervical
adenocarcinomas rarely do so. Endometrial adenocarcinoma also rarely metastasizes to
the lung. Its tumor cells express CA 125. A metastatic low-grade endometrial stromal

73
sarcoma shows in TTFNA abundant single and clustered small round cells with scant
cytoplasm. A metastatic myometrial leiomyosarcoma to the lung shows malignant
spindle cells with elongated unclei with blunt ends.

Ovary
Ovarian carcinoma metastatic to the lung frequently involves the pleura with associated
malignant effusion. Parenchymal pread is uncommon and occurs late in the disease.
Cells derived from an ovarian adenocarcinoma usually express CA125, vimentin and
estrogen receptor and they are CEA negative.

Skin
Cutaneous melanoma frequently spreads to the lung and commonly yields single
pleomorphic malignant cells. Intranuclear cytoplasmic inclusions are commonly seen
and intracytoplasmic melanin pigment granules may be noted. The tumor cell cytoplasm
characteristically expresses S100 protein, HMB-45, MART1 and melan A (Fig. 5.11).

74
Fig. 5.11. Metastatic cutaneous melanoma to the lung showing in TTFNA pleomorphic
dyshesive malignant cells that stain positively with HMB-45 antibody. (A: Pap, x 500; B:
ABC, x 500).

Soft tissue and bone tumors

Soft tissue sarcomas commonly spread to the lung. Bone sarcomas rarely metastasize
to the lung with the exception of Ewing sarcoma. Metastatic deposits of soft tissue
sarcomas in the lung are usually parenchymal and are most commonly diagnosed by
TTFNA. In clinical practice, the presence of malignant nonepithelial cells in a pulmonary
cell sample from a patient with a known soft tissue or bone sarcoma is often diagnostic
of metastatic sarcoma.

Testicular and extragonadal germ cell tumors

Testicular seminomas rarely spread to the lung. Other gonadal tumors often
metastasize to the lung. Cells derived from a nonseminomatous tumor deposits are
usually pleomorphic and occur singly or in syncytial clusters. The tumor cell cytoplasm
expresses alpha-fetoprotein and placental alkaline phosphatase. Cells from a
choriocarcinoma express beta human chorionic gonadotropin.

Neuroendocrine carcinomas
Neuroendocrine cancer arising from extrapulmonary locations (gastrointestinal tract,
pancreas and ovary) may spread to the lung. These tumors yield single and clustered
epithelial cells with eccentrically located nuclei (plasmacytoid configuration) and
chromatin clumping. A positive cytoplasmic reaction with neuron-specific enolase,
synaphtophysin, chromogranin and CD56 antibodies will confirm the diagnosis.
However, determination of the location of the primary tumor cannot be made with
confidence on cytologic bases alone.

Lymphoma and leukemia

Hodgkin disease, Non-Hodgkin lymphoma and leukemias commonly spread to the lung.
The metastatic tumor cells in different pulmonary cell samples are similar to those of
the primary neoplasms seen in blood, bone marrow needle aspirate and lymph node
FNA. Flow cytometry and/or Immunocytochemistry may be used to subtype metastatic
Non-Hodgkin lymphoma.

BIBLIOGRAPHY

Colby TV, et al. Tumors of the lower respiratory tract. In Atlas of tumor pathology,
Washington DC, Armed Forces Institute of Pathology, 1995.

Dabbs DJ. Immunohistology of metastatic carcinoma of unknown primary. In Diagnostic

Immunhistochemistry, D. Dabbs, ed, 2nd ed, 2006, Philadelphia, Churchill Livingstone
Elsevier, p: 180.

75
DeLellis RA, Hoda RS. Immunohistochemistry and molecular biology in cytological
diagnosis. In Koss’ Diagnostic cytology and its histopathologic bases. 5th ed, Koss LG,
Melamed MR, eds. Philadelphia, Lippincott Williams & Wilkins. 2006, p 1635.

Erozan YS. Cytopathology in pulmonary biopsy procedures. In Biopsy techniques in

Pulmonary Disorders, Wang KP, ed. New York, Raven Press, 1989, p 139.

Flint A, Lloyd RV. Colon carcinoma metastatic to the lung: cytologic manifestations and
distinction from primary pulmonary adenocarcinoma. Acta Cytol. 1992; 36; 230.

Koss LG, Melamed MR. Tumors of the lung: conventional cytology and aspiration
biopsy. In Koss’ Diagnostic cytology and its histopathologic bases, 5th ed, Koss LG,
Melamed MR, eds. Philadelphia, Lippincott Williams & Wilkins. 2006, p 643.

Nguyen GK, Berendt R. Aspiration cytology of metastatic endometrial stromal sacoma,

adenosarcoma of the uterus and mixed mesodermal tumor. Diagn Cytopathol. 1986; 2:
256.

Nguyen GK. Fine needle aspiration biopsy cytology of metastatic renal cell carcinoma.
Acta Cytol. 1988; 32;409.

Nguyen GK, et al. Transmucosal needle aspiration biopsy via fiberoptic bronchoscope.
Value and limitations in the cytodiagnosis of tumors and tumor-like lesions of the lung.
Pathol Annu. 1992; 27(1): 105.

Nguyen GK, Kline TS. Essentials of cytology. An atlas. New York, Igaku-Shoin, 1993, p
43.

Park SY, et al. Panels of immunohistochemical markers help determine primary sites of
metastatic adenocarcinoma. Arch Pathol Lab Med. 2007; 131: 1561.

Raab SS, et al. Metastatic tumors in the lung: a practical approach to diagnosis. In
Practical pulmonary pathology. Leslie KO and Wick WR, eds. Philadelphia, Churchill
Livingstone, 2005, 603.

Tao LC. Lung, pleura and mediastinum. In Guides to clinical aspiration biopsy, Kline TS,
ed. New York, Igaku-Shoin, 1988.

Travis WD, et al. Pathology and genetics of tumours of the lung, pleura, thymus and
heart. In WHO classification of Tumours, Lyon, IARCPress, 2004.

76
Chapter 6

PLEURAL TUMORS
Pleural tumors can be malignant or benign. Important malignant tumors of the pleura
consist of diffuse malignant mesothelioma (commonly known as mesothelioma),
primary effusion lymphoma and metastatic cancers. Benign pleural neoplasms are very
rare. Pleural cancers are commonly associated with pleural serous effusions that often
contain exfoliated malignant cells.

A. MESOTHELIOMA

Pleural mesothelioma is an aggressive cancer. Its incidence of varies among different

surveyed populations, and incidences of 0.65-21.4 cases in men and 0.35-1.9 cases in
women per million population per year have been reported from different countries,
and from different states in the United States. Epidemiologic studies have linked
occupational exposure to asbestos to the development of pleural mesotheliomas in 70
to 90% of the cases and the average latent period is 35 years. Other etiologic factors
include exposure to erionite, therapeutic radiation and chronic infection. The tumor
occurs mainly during the fifth and sixth decades of life and rarely in children. Males
comprise 75% of all reported cases, and almost all patients with the disease die within
6 to 12 months after the diagnosis. It may present as a diffuse or localized growth, and
the diffuse form accounts for about 75% of all cases. In over 90% of patients the
disease manifests initially with recurrent, unilateral, bloody pleural effusions. In less
than 10% of the cases, pleural tumors without pleural effusions are detected by chest
radiography. Thus, pleural mesotheliomas are more commonly evaluated by cytologic
examination of associated effusions.

COLLECTION AND PREPARATION OF CELL SAMPLES

Proper collection and preparation of cytologic specimens are critical for an accurate
diagnosis of mesothelioma. The preparation techniques vary with the types of cell
samples.

Pleural effusion. The diagnosis of mesothelioma by effusion cytology depends largely

on the number of tumor cells present in the specimen. For optimal results the whole
effusion sample should be submitted for cytologic evaluation and no fixative is needed if
the specimen is prepared without delay. Effusion samples without added fixatives kept
in the refrigerator at 40 C will preserve the cell morphology for several days. The
minimum amount of a fluid sample that commonly yields adequate cells for
cytodiagnosis is about 200 mL.

77
Depending on the amount of blood present, the preparation techniques are different.
For a non-bloody sample, usually 4 cytospin smears or preparations and a cell block are
prepared by cytocentrifuge technique. For examination by electron microscopy (EM), a
small portion of the sediment is fixed in a vial of 2% glutaraldehyde. It should be borne
in mind that ethanol is not a suitable fixative for EM study as it destroys the
ultrastructure of the fixed cells. Immunocytochemical (IM) staining with commercial
antibodies can be done on Papanicolaou stained smears without prior destaining and on
cell block sections, using the routine avidin-biotin-complex technique.

A blood-stained effusion requires special preparation to obtain satisfactory smears.

Minimal blood contamination may be overcome by fixing smears in Carnoy solution for 3
to 5 minutes to lyse red blood cells. If the fluid sample is heavily contaminated with
blood, it should be mixed with an equal volume of a density gradient solution such as
Ficoll-Hypaque. The mixture is subsequently centrifuged to separate nucleated cells
from red blood cells, and the layer of nucleated cells is then removed with a pipette for
preparation of smears and a cell block.

Fine needle aspirate. Cells from a pleural mass lesion can be sampled by TTFNA.
Usually a disposable 22-gauge and 15-cm-long spinal-type needle is used. Several direct
smears are prepared from the needle aspirate. They can be fixed in 95% ethanol for
staining with the Papanicolaou method or with hematoxylin and eosin, or they can be
air-dried for staining by the Diff-Quik technique. Minute tissue fragments retrieved
from the needle aspirate should be fixed in 2% glutaraldehyde for EM study. The
needle and syringe can be rinsed in a vial of a salt-balanced solution to be used for cell
block preparation.

HISTOLOGY, IMMUNOHISTOCHEMISTRY AND ULTRASTRUCTURE

Mesotheliomas may be classified into 4 main histologic types: epithelial, sarcomatous,
desmoplastic and biphasic or mixed mesotheliomas. About 50% of pleural
mesotheliomas are of epithelial type and consist of polygonal epithelial cells with
variable degrees of atypia/anaplasia arranged in tubulopapillary, microcystic or solid
pattern. Usually, 2 or 3 histologic patterns coexist in almost all epithelial mesotheliomas.
(Fig. 6.1). Sarcomatous and mixed mesotheliomas account for approximately 15 - 20%
and 25 - 30% of all cases, respectively. A sarcomatous mesothelioma is characterized
by spindle malignant cells arranged in a non-specific pattern. A mixed mesothelioma is
composed of epithelial and sarcomatous elements, and areas showing a transition
between these 2 types of cellular element may be seen. (Fig. 6.1). Epithelial and mixed
mesotheliomas are commonly associated with pleural effusions that usually contain
exfoliated epithelial tumor cells. In contrast, sarcomatous tumors are rarely associated
with pleural effusions, and when they do, they seldom exfoliate their cells in the
effusions.

78
In tissue sections, an epithelial mesothelioma show several IM characteristic features:
- Negative cytoplasmic reactions to epithelial antibodies such as CEA, B72.3, MOC31
antibodies.
- Cell membrane positive reactions with EMA, HBME-1, thrombomodulin and mesothelin
antibodies.
- Positive cytoplasmic reaction to pan-cytokeratin, vimentin and cytokeratins 5/6
antibodies.
- Positive cytoplasmic/nuclear reaction to calretinin antibody.
- Positive nuclear reaction to Wilms tumor gene product 1 (WT1) antibody. (Fig. 6-2).

Sarcomatous mesothelioma cells show positive cytoplasmic reactions to vimentin and

cytokeratin antibodies. They may also express calretinin, desmin and actin.

By electron microscopy, cells of an epithelial mesothelioma are characterized by well-

formed desmosomes, long filamentous microvilli with length: diameter ratio > 12 to 15
are present on the free cell surfaces and intracytoplasmic bundles of intermediate
filaments. (Fig. 6.3). Cells of a sarcomatous mesothelioma are spindle-shaped and differ
very little from fibroblasts, and aborted microvilli may rarely be observed on the cell
surfaces. A mixed mesothelioma shows tumor cells with features of both epithelial and
sarcomatous mesotheliomas, and a transition between the two above-mentioned types
of cell may be observed.

79
 B

C
Fig. 6.1. Histology of different types of pleural mesotheliomas:
A. Epithelial mesothelioma consisting of polygonal tumor cells in solid and glandular
patterns.
B. Sarcomatous mesothelioma consisting of spindle tumor cells in a nonspecific pattern.
C. Mixed mesothelioma showing a mixture of epithelial and sarcomatous cells.
(HE, x 250).

80
Fig. 6.2. Pleural epithelial mesothelioma showing tumor cells with positive nuclear
staining to WT1 antibody. (ABC, x 250).

Fig. 6.3. Ultrastructure of an epithelial mesothelioma showing tumor cells with well-
formed desmosomes and long filamentous microvilli without dense-core rootlets.
(Uranyl acetate and lead citrate, x 36,000).

EFFUSION CYTOLOGY
Epithelial and Mixed Mesotheliomas. Serous effusions associated with an
epithelial or mixed mesothelioma are usually hypercellular and contain numerous
epithelial tumor cells. About 10% of effusions associated with pleural mesotheliomas
are acellular or extremely scanty in cellularity, and about 50% of patients have
cytologically negative pleural effusions. In industrialized country about 1% of
malignant pleural effusions are cause by diffuse malignant mesothelioma. A more
comprehensive discussion on effusion cytology of mesothelioma may be found in
chapter 1 of the author’s monograph on Essentials of Fluid Cytology.

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Mesothelioma cells in effusions are almost always of epithelial type, as sarcomatous
tumor cells seldom exfoliate into the fluid. The first detailed description of effusion
cytology of mesotheliomas was reported by Klempman in 1961. Since then
numerous publications on this topic have appeared in the literature. The cytologic
manifestations of epithelial mesotheliomas have been summarized by DeMay as
follows: “a more and bigger cells in more and bigger clusters, and morphologic
kinship with mesothelial cells”. The large ball-like clusters of tumor cells may show
knobby, lobulated or smooth borders and consist of a dozen to a few hundreds cells.
Admixed with these large cell balls are single and clustered tumor cells. Papillary
tumor tissue fragments may be seen and are regarded by some investigators as a
characteristic feature of epithelial mesothelioma.

In about 50% of the cases, the tumor cell clusters and single neoplastic cells are
roughly equal in numbers, and in the remaining cases either cell clusters or single
cells predominate. The tumor cells that are present singly and in small clusters may
show different degrees of nuclear atypia ranging from pleomorphic to bland and
frequently lack the significant atypia present in carcinoma. Their cytoplasm may
display features of mesothelial cells with ecto-endoplasmic demarcation and fuzzy
border around the entire perimeter. A “two-tone” cytoplasmic staining with pink-
orange endoplasm and blue-green ectoplasm may be observed in tumor cells
stained with the Papanicolaou method. Long slender microvilli may be seen on the
free surfaces of tumor cells exfoliated from a well-differentiated epithelial
mesothelioma. Very large or giant mesothelial cells may be noted. Intercellular clear
spaces or windows and cell arrangements with configurations such as “cell-
embracing-cell”, “pincer-like grip” and “cell-in-cell” or “cell engulfment” are
commonly found in small tumor cell clusters. Irregular or papillary tumor tissue
fragments with fibrovascular cores may be observed in some cases. (Fig. 6.4 to
Fig. 6.9).

These cellular arrangements are non-specific for epithelial mesothelioma as they

may also be seen in a serous effusion secondary to a metastatic adenocarcinoma.
Occasional tumor cells with signet-ring configuration are observed. Numerous
mesothelial cells with normal and atypical nuclei are almost always present.
Intracytoplasmic vacuoles or blebs located at the periphery of the tumor cell
cytoplasm or in the paranuclear area are best visualized in air-dried smears stained
with the Diff-Quik or May-Grunwald-Giemsa technique.

IM staining of the effusion cell block sections with selected commercially available
proves to be useful in confirming tumor cell lineage.

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 A

83
Fig. 6.4. Serous effusion in a pleural epithelial mesothelioma of showing in:
A and B. Tumor cells present singly and in ball-like and smaller clusters. Abundant,
granular cytoplasm and cell-embracing-cell arrangement are observed.
C. Clear space or “window” between two adjacent cells and “two-tone” cytoplasm
are noted. (Pap, A x 100, B and C x 400).

Fig. 6.5. A thick papillary tumor tissue fragment with fibrovascular core is seen in
associated effusion of a case of pleural epithelial mesothelioma. (Pap, x 100).

Fig. 6.6. Pleural effusion in a case of epithelial mesothelioma showing tumor cells
present predominantly in large tridimensional ball-like clusters. (Pap, x 250)

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Fig. 6.7. Pleural epithelial mesothelioma showing in associated effusion malignant
cells present singly or in small loose clusters. (Pap, x 500).

Fig. 6.8. Pleural epithelial mesothelioma showing single and small, loose clusters
of tumor cells with large intracytoplasmic vacuoles or “signet-ring” configuration.
(Pap, x 500).

As the cytologic manifestations in serous effusions of an epithelial or mixed

mesotheliomas mimic or overlap with those of a metastatic adenocarcinoma in the
majority of cases, IM studies of the effusion cell block with selected antibodies or EM
examination of effusion sediment are necessary for final diagnosis.

Cell Block (CB). Sections may show epithelial tumor cells in solid, acinar and
papillary patterns with fibrovascular or hyalinized fibrous cores reflecting papillary
tumor fragments broken from the main tumor mass. (Fig. 6.9). The acinar clusters
of tumor cells with central hyalinized fibrous cores are of diagnostic value for
epithelial mesothelioma, according to Whitaker.
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Fig. 6.9. Pleural effusion CB from an epithelial mesothelioma shows tissue fragments with
fibrovascular cores and tumor cell clusters. (HE, x 250).

Cytochemistry and Immunocytochemistry. Cells of an epithelial or mixed mesothelioma

are rich in glycogen, and stain positively with periodic acid Schiff. These cells usually
express high- and low-molecular weight cytokeratins and vimentin and stain negatively
with epithelial antibodies such as CEA, B72.3, BG8, Ber-Ep4, MOC31 and Leu-M1
antibodies. Mesothelioma cells often stain positively with HBME-1, calretinin,
thrombomodulin, CK 5/6, mesothelin and WT1 antibodies. (Fig. 6.10 to Fig. 6.12). Among
those positive markers calretinin, CK 5/6 and WT1 are currently the best positive makers
of epithelial mesothelioma.

Positive immunostaining reactions of epithelial mesothelioma cells with the above-

mentioned mesothelial antibodies display important characteristic features:

A. A cell-membrane staining pattern is observed with HBME-1, thrombomodulin and

mesothelin antibodies.
B. A cytoplasmic staining pattern is noted with CK 5/6 antibodies.
C. A cytoplasmic and nuclear staining pattern is observed with calretinin antibody.
D. A nuclear staining pattern is observed with WT1 antibody.

According to Ordonez a combination of 2 positive (calretinin, CK 5/6, WT1) and 2

negative markers (CEA, B72.3, MOC31) is adequate for a firm diagnosis of epithelial
mesothelioma. Battifora requires a negative reaction with 3 epithelial antibodies and a
positive reaction with 3 mesothelial-related antibodies of the tumor cells for a positive
diagnosis of epithelial mesothelioma. If IM studies give equivocal or inconclusive results
electron microscopic study of the tumor cells (cell block) is needed for further
confirmation.
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 A

C
Fig. 6.10. Pleural effusion CB from an epithelial mesothelioma showing:
A. Tumor cells with negative cytoplasmic reaction to CEA antibody.
B. Tumor cells with positive cytoplasmic reaction to calretinin antibody.
C. Tumor cells displaying a strong and positive, “thick” membranous reaction with

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spiking pattern to EMA antibody. The spiking pattern reflects the presence of long,
slender microvilli present on the outer aspects of some tumor cell clusters. (ABC, x 250).

Electron microscopy. Ultrathin sections from the effusion cell block stained with
uranyl acetate and lead citrate commonly reveal polygonal, non-mucus secreting
epithelial-like cells with well-formed cell junctions, abundant intracytoplasmic
glycogen granules, perinuclear bundles of intermediate filaments and long slender
microvilli without dense-core rootlets on the free cell surfaces. Long microvilli with a
length/diameter ratio greater than 12 or 15 are a characteristic feature of an
epithelial mesothelioma (Fig. 6.11).

Fig. 6.11. Ultrastucture of an effusion CB from a pleural mesothelioma showing

tumor cells with long filamentous microvilli on the tumor cell membrane. (Uranyl
acetate and lead citrate, x 24, 000).

Cells exfoliated from an epithelial mesothelioma with marked cellular atypia or anaplasia
may show no distinctive cytologic, IM and ultrastructural features.

FNA CYTOLOGY
Only a small number of pleural mesotheliomas with cytologic evaluation by TTFNA
have been reported. An epithelial mesothelioma usually yields tumor cells singly, in
thick clusters, in sheets and in ball-like or papillary clusters. The individual tumor
cells display well-defined, optically dense cytoplasm, oval nuclei and prominent
nucleoli. Tumor cells showing vacuolated cytoplasm may be observed. (Fig. 6.12 and
Fig. 6.13). However, an epithelial mesothelioma consisting of anaplastic tumor cells
may yield large malignant cells with ill-defined or well-defined, granular cytoplasm
and prominent nucleoli singly and in aggregates, similar to those of a large cell
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carcinoma. TTFNA from a sarcomatous tumor may reveal spindle malignant cells
with elongated nuclei and scant, granular or clear cytoplasm present singly and in
loose clusters (Fig. 6.14). A mixed mesothelioma is characterized by an admixture of
single and clustered malignant spindle cells and malignant epithelial cells displaying
mesothelial cell features.

B
Fig. 6.12. Pleural epithelial mesothelioma showing in TTFNA:
A. Ball-like and papillary tumor cell clusters or fragment.
B. Tumor cells with prominent nucleoli present in tridimensional clusters and singly.
(Diff-Quik, A x 100, B x 500).

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Fig. 6.13. Epithelial mesothelioma showing in TTFNA tumor cells singly, in a loose
sheet and in a tridimensional cluster. (HE, x 500).

Fig. 6.14. Sarcomatous mesotheliomas of showing in TTFNA isolated, monomorphic

spindle cells with elongated nuclei and scanty cytoplasm. (Pap, x 500).

Immunostaining of the tumor cells within the cell block or aspirated minute tissue
fragments with antibodies against CEA, Ber-Ep4, MOC-31, CK 5/6, calretinin and
WT1 are helpful for further tumor typing (please see page 86).

By EM the epithelial tumor cells show well-formed desmosomes and long slender
microvilli. (Fig. 6.15). Microvilli in direct contact with collagen fiber bundles in the
tumor matrix may be seen in minute tumor tissue fragments, and this finding
constitutes a strong evidence indicating an invasive epithelial mesothelioma,
according to Ghadially.

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Fig. 6.15. Ultrastucture of an aspirated minute tumor tissue fragment from a
pleural epithelial mesothelioma showing tumor cells with well-formed cell junctions
and long, filamentous microvilli on the tumor cell surface. (Uranyl acetate and lead
citrate, x 24,000).

DIAGNOSTIC ACCURACY
According to Whitaker a diagnosis of epithelial mesothelioma by effusion cytology
may be suggested in the presence of many large clusters or aggregates of tumor
cells together with abundant single neoplastic cells. Large cellular aggregates are
of most value in facilitating the diagnosis of malignancy, and single cells or clusters of 2
to 6 cells are of most value in identifying the mesothelial characteristics of tumor cells.
Nuclear atypia, as commonly seen in metastatic adenocarcinoma, are not usually seen in
mesothelioma cases. IM and/or EM studies the effusion cell block are necessary for
distinguishing an epithelial mesothelioma from an adenocarcinoma. In the experience of
Whitaker, et al. a diagnostic accuracy rate of 80% of mesotheliomas of has been reached
by a combination of effusion cytology and IM and/or EM studies of effusion cell blocks.
The predictive value of a positive diagnosis of epithelial mesothelioma in serous effusion
has been about 100% in those investigators’ hands. For FNA diagnosis of pleural
mesothelioma a sensitivity rate of 73-78% has been reported.

DIFFERENTIAL DIAGNOSIS
The cytologic differential diagnosis between markedly reactive mesothelial cells and
epithelial mesothelioma cells can be problematic. The tumor cell nuclei may stain
positively with p53 antibody, while reactive mesothelial cells show no staining with this
antibody. Estimation of the proliferative fraction using antibody to Ki-67 (MIB1) may be
helpful in the distinction of benign from malignant effusions, as malignant effusions
have a Ki-67 immunostain labeling index value > 20% in 82% of cases while the index
value of benign effusion is <5%. Ploidy determination by flow cytometry or cell image
analysis is not helpful in solving this diagnostic dilemma as 50 to 85% of epithelial
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mesotheliomas are diploid. Some investigators have claimed that reactive mesothelial
cells do not express EMA and that mesothelioma cells show a strong immuno-positive
reaction with this antibody. However, this finding was not supported by the work of
others. In practice, if a cytodiagnosis of epithelial mesothelioma is uncertain, tissue
biopsy of the pleural lesion should be done for histologic, IM and EM studies.

Spindle cell sarcomas of the lung and chest wall. Malignant spindle cells derived
from a sarcomatous mesothelioma should be differentiated from those of a benign
solitary fibrous tumor of the pleura, and from those of a fibrosarcoma, leiomyosarcoma,
malignant schwannoma and malignant fibrous histiocytoma of the lung and pleura. IM
staining of the needle aspirate with cytokeratin antibody is helpful in this situation as
sarcomatous mesothelioma cells express cytokeratin while those of the other above-
mentioned 5 tumors do not. Cells from a benign solitary fibrous tumor of the pleura
commonly express CD34 and react negatively to cytokeratins. A carcinosarcoma of the
lung and a primary or metastatic synovial sarcoma of the lung and pleura should be
considered in the differential diagnosis with a mixed mesothelioma in cytologic material
obtained by FNA. Epithelial and sarcomatous cells aspirated from a mixed mesothelioma
are cytokeratin positive and CEA negative. Epithelial and sarcomatous cells from a
carcinosarcoma of the lung may express CEA. Tumor cells from a biphasic synovial
sarcoma stain positively with cytokeratin and vimentin antibodies, and the epithelial
component of the tumor may express, in addition, CEA. EM study of synovial sarcoma
cells is not helpful as the neoplastic epithelial cells may show long and slender microvilli
similar to those seen in the epithelial component of a mixed mesothelioma, according to
Ghadially.

B. SECONDARY CANCERS

Pleural metastasis usually occurs in the context of lymphangitic or vascular spreading

of cancer cells. Cancers may also metastasize to the pleura by retrograde lymphagitic
spread or by direct invasion through the diaphragm. Abdominal cancers that
metastasize to the pleura usually involve the liver. Cancers arising from the lung,
stomach, breast and ovary more commonly metastasize to the pleura. Metastatic
cancers to the pleura are commonly associated with a positive pleural effusion.
Effusion cell samples in these cases are prepared by the same method described in
the section of pleural mesothelioma. The reader is referred to chapter 1 in the
author’s monograph on Essentials of Fluid Cytology for a more comprehensive
discussion on metastatic cancers to the pleura.

Malignant Epithelial Tumors

Metastatic squamous cell carcinoma rarely exfoliates its cells in associated
effusions. The cytologic manifestations of a well-and a poorly differentiated squamous
cell carcinoma are different. Cells derived from a well-differentiated tumor occur singly
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or in dyshesive clusters and show a well-defined, “hard”, orangeophilic or basophilic
cytoplasm with a thick cytoplasmic rim and hyperchromatic, pleomorphic nuclei (Fig.
6.16). A poorly differentiated tumor exfoliates its cells in syncytial clusters with ill-
defined cytoplasm and oval or pleomorphic, hyperchromatic nuclei. (Fig. 6.17).

Fig. 6.16. Metastatic keratinizing squamous cell carcinoma to the pleura shows in
associated effusion keratinizing malignant cells. (Pap, x 500).

Fig. 6.17. Effusion secondary to a metastatic non-keratinizing squamous cell

carcinoma to the pleura shows tumor cells in cohesive sheets or clusters. (Pap, x
250).

Metastatic adenocarcinomas account for over 90% of malignant pleural effusions.

The exfoliated cancer cells, regardless of their primary tumors usually occur in tri-
dimensional clusters of varying sizes with smooth contours, in small clusters and
singly. The tumor cells may display a vacuolated cytoplasm, eccentrically located

93
nuclei and prominent nucleoli. (Fig. 6.18). A few additional cytologic findings may be
seen in cell groups derived from adenocarcinomas arising from certain anatomic sites.
Psammoma bodies may be seen in effusions associated with a metastatic ovarian
serous cystadenocarcinoma, papillary adenocarcinoma of the lung and papillary
carcinoma of the thyroid. A metastatic mammary duct carcinoma exfoliates small
cancer cells with linear arrangement or “Indian files” pattern. A metastatic gastric
signet-ring cell carcinoma exfoliates single cancer cells with large cytoplasmic vacuole
and eccentrically located oval nuclei with prominent nucleoli. (Fig. 6.19). A metastatic
mucinous adenocarcinoma shows groups of cancer cells with vacuolated cytoplasm in
a mucous background. Metastatic adenocarcinoma cells usually react positively with
CEA, Ber-Ep4, Leu M1 and MOC31 antibodies. (Fig. 6.20). Tumor cells derived from a
bronchogenic carcinoma are commonly TTF1 positive.

B
Fig. 6.18. Metastatic adenocarcinoma to the pleura showing in effusion:
A. Tumor cells in large tridimensional ball-like clusters, in small clusters and singly.
B. Tumor cells with “cell-embracing-cell” arrangement. (Pap, A x 100, B x 500)
94
Fig. 6.19. Metastatic signet-ring cell adenocarcinoma showing in effusion single
malignant cells with some displaying intracytoplasmic vacuoles. (Pap,x 500).

95
 B

C
Fig. 6.20. CB section from a pleural effusion secondary to a metastatic bronchogenic
adenocarcinoma showing tumor cells displaying positive cytoplasmic reaction to CEA
(A), positive membranous reaction to MOC31 (B) and positive nuclear reaction to
TTF1 (C) antibodies. (ABC, x 250).

Metastatic bronchogenic small cell carcinoma exfoliates small malignant cells

with round nuclei showing a salt and pepper chromatin pattern and scant cytoplasm
in small clusters displaying nuclear molding. (Fig. 5.21 and Fig. 6.22). Micronucleoli
may be present.

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Fig. 6.21. Metastatic small-cell carcinoma to the pleura shows in associated effusion a
loose cluster of small malignant cells displaying focal nuclear molding. (Pap, x 400).

Fig. 6.22. Metastatic small-cell carcinoma to the pleura shows in effusion a tight
cluster of tumor cells with molding. (Pap, x 500).

Metastatic large-cell carcinoma yields single and loosely clustered large malignant
cells with abundant, well-defined cytoplasm, enlarged hyperchromatic nuclei and
prominent nucleoli. (Fig. 6.23).

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Fig. 6.23. Metastatic large-cell carcinoma to the pleura shows in associated effusion
large pleomorphic malignant cells present predominantly singly. (Diff-Quik, x 500).

Metastatic neuroendocrine carcinoma shows dyshesive cell clusters with

regular, round nuclei with chromatin clumping and conspicuous nucleoli. A positive
cytoplasmic reaction with neuron-specific enolase or chromogranin antibody will
confirm its neuroendocrine differentiation.

Non-Epithelial Cancers
Lymphoma and Leukemia are the most common non-epithelial cancers associated
with pleural effusions. A low-grade lymphoma or a lymphocytic leukemia exfoliates
monomorphic benign-appearing lymphoid cells similar to mature lymphocytes. A high-
grade lymphoma exfoliates monomorphic lymphoid cells with nuclear protrusion and
indentation. (Fig. 6.24). Reed-Sternberg cells may be seen in serous effusion
secondary to Hodgkin disease. A chronic myelogenous leukemia shows mature and
immature myelogenous cells without “leukemic hiatus”. A multiple myeloma yields
malignant plasma cells with bizarre forms.

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Fig. 6.24. Non-Hodgkin lymphoma involving the pleura showing in pleural effusion
monomorphic malignant lymphoid cells. Some tumor cells display nuclear
indentation or protrusion. (Pap, x 500).

A metastatic melanoma shows single and clustered malignant cells with some
dysplaying intranuclear cytoplasmic inclusions and prominent nucleoli.
Intracytoplasmic melanin pigment granules may be present. A positive cytoplasmic
reaction with HMB-45 or MART 1 antibody will confirm their melanocytic
differentiation.

A metastatic neuroblastoma yields small round cells with scant and fibrillary
cytoplasm and tumor cells forming rosettes may be observed. The tumor cell
cytoplasm stains positively with neuron-specific enolase and chromogranin antibodies.

Bone and Soft Tissue Sarcomas show single and loosely clustered cancer cells.
The tumor cells usually loose their original shapes as seen in tumor tissue sections
and tend to have a round configuration. Tumor typing is difficult without clinical data
and immunocytochemical studies. Some tumors may show specific cytologic findings.
A metastatic chondrosarcoma yields single chrondrocyte-like cells with abundant,
well-defined cytoplasm and prominent nucleoli. (Fig. 6.25). A metastatic Ewing
sarcoma yields loosely clustered polygonal cells with oval nuclei and moderate
amount of cytoplasm that stains positively with periodic acid-Schiff reagent. A
metastatic biphasic synovial sarcoma shows small epithelial-like cells with oval
nuclei and scant cytoplasm in clusters with focal gland-like arrangement and spindle
cells with scant and ill-defined cytoplasm and oval nuclei. Cellular changes suggesting
the transformation of spindle cells to epithelial-like cells have been observed. The
tumor cells usually express cytokeratin and vimentin and stain negatively with CEA
antibody.

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Fig. 6.25. Metastatic chondrosarcoma showing in associated effusion single malignant
cells with conspicuous nuclei and ill-defined basophilic cytoplasm. (Pap, x 500).

Diagnostic Accuracy. Effusion cytology is more sensitive than blind biopsy of the
pleura in detecting pleural cancer (80% versus 45%) in experienced hands. False-
negative diagnostic rates vary widely among reported series. The main reasons are
inadequate cell sample, scantiness of malignant cells, faulty preparatory techniques
and erroneous interpretations. False-positive diagnostic rates up to 3% have been
reported. The most common error is the misinterpretation of highly reactive
mesothelial cells in long-standing benign effusions as malignant glandular cells.

D. OTHER PLEURAL TUMORS

Benign localized epithelial mesothelioma or adenomatoid tumor is an

exceedingly rare neoplasm of the pleura. It is small and incidentally found in the lung
resected for other conditions.

Solitary fibrous tumor is a rare lesion that is most likely arising from the subpleural
mesenchymal cells. It is not related to asbestos exposure and has no sex predilection.
The tumor is asymptomatic and usually discovered incidentally by chest
roentgenograms and it measures up to several cm in greatest dimension. The tumor
is well-demarcated with pushing borders and is often pedonculated. Most solitary
fibrous tumors of the pleura are benign but about 30% are malignant. About 80% of
these tumors arise from the visceral pleura and the remainder originates from the
parietal pleura. Histologically, it is characterized by benign fibroblastic cells arranged
in a non-specific pattern and a collagenous, hyalinized stroma. Cellular atypia and
mitoses are uncommon. The tumor yields in TTFNA a scanty cellular material showing
bland spindle cells that react positively with CD34 and negatively with cytokeratin
antibodies, in contrast to those of a fibrous mesothelioma. (Fig. 6.26).
100
Fig. 6.26. TTFNA from a localized fibrous tumor of the pleura shows benign,
spindle cells with elongated nuclei in no specific pattern. (Pap, x 500).

Primary effusion lymphoma is a highly aggressive neoplasm of large B-cell

presenting as a serous effusion without detectable tumor masses. It is associated with
human herpes virus 8(HHV8)/Kaposi sarcoma herpes virus (KSHV) and usually occurs
in HIV-positive patients. The most common sites of tumor involvement are pleural,
peritoneal and pericardial serosal cavities. Cytologically, the tumor cells in the effusion
consist of dispersed large pleomorphic cells or large lymphoid cells with immunoblastic
features. The reader is referred to Chapter 1 in the author’s monograph on Essentials
of Fluid Cytology for more discussion.

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THE END

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