Endobronchial Ultrasound (EBUS) guided FNA

It is ​FNA ​performed through the ​bronchial wall ​using a ​bronchoscope ​and real-time ​ultrasound ​guidance.

LUNG CYTOLOGY
Schmitt FC. Cytopathology 2011

LUNG CYTOLOGY
• ​Diagnosis of lung cancer
• ​Detection of infections
• ​Evaluation of interstitial diseases.
• ​The importance of cytological techniques for investigation of respiratory conditions has been recognized since the earliest days of clinical cytology.
• ​The last few decades have shown a clear demonstration of the sensitivity and predictive value of cytodiagnosis of lung tumors and an acceptance of
all cytological modalities as a basis for management.
• ​Nowadays, in around 40% of lung cancer cases, the only material available for diagnosis and molecular testing is the cytological material.
LUNG CYTOLOGY Sensitivity
SAMPLING LOCATION OF THE LESION
CENTRAL PERIPHERAL
SPUTUM 70-85% 30-60% BRONCHIAL
70-90% 61-76% WASHING BRONCHIAL BRUSHING
Gray and Kocjan, 2010; Bibbo M, 2008; Layfield et al, 1996; Rosenthal DL, 1988

77-90% 50-70%
BAL 80-90% 70-80% FNA 80-95% 80-95%
Department of Pathology and Oncology, Medical Faculty of Porto University
General
President of the International Society of Breast Pathology

LUNG CYTOLOGY EBUS

President of the Portuguese Society of Cytology
Head of Molecular Pathology Unit,
-
Secretary of the International Academy of
Prof. Fernando Schmitt
IPATIMUP
Cytology
No financial disclosures

27/08/2019​EBUS-FNA Indications
• ​Staging and restaging of ​lung cancer
• ​Staging of ​extrathoracic ​malignancies
• ​Diagnosis of ​granulomatous ​disease (eg. sarcoidosis and mycobacterial infection)
• ​Evaluation of mediastinal lymphadenopathies and masses of ​undetermined ​aetiology

1
•​Technique:
•​Identify targeted area

•​Inserted needle under real- ​time US guidance

•​Stab the target 10–15 times ​without suction and apply suction only for the last two or three stabbing motions

•​Specimen air-flushed on a ​slide

•​Needle flushed with heparin- ​saline

• Procedure room: ​endoscopy or operating room
• Anesthesia​: conscious sedation or under general anesthesia
• Ultrasound
– ​RADIAL EBUS ​provide a 360 view of the airway wall and surrounding ​structures ​– ​LINEAR EBUS ​provides a wedge-shaped image of the airway wall and ​adjacent structures

• US image​: can be optimized with a water-filled balloon at the US ​probe-airway wall interface.
• Colour Doppler​: allows easy identification of ​vascular structures​.

EBUS-FNA procedure
• ​A single needle pass​: defined as a single insertion of the aspiration needle, from entry to exit, through
the airway wall into the target site
• ​includes 5 to 15 excursions of the needle within ​the target lesion.
​ iagnosis and staging: minimum of 3 passes.
• ​Lung cancer d
 EBUS-FNA Number of Passes
Rapid on-site evaluation (ROSE)
EBUS-FNA Sampling Errors ​
• ​Is to examine the sample during the procedure, in
• ​If the target is ​not appropriately identified ​and correlated
general using a ​rapid stain. ​with CT findings
​ ontaminated ​with airway epithelium or blood.
• ​Non-diagnostic samples are generally c
• ​Evaluated by a ​cytopathologist ​or a trained ​cytotechnologist
​ alse-negative rates of up to 15–20% can be seen
• F
• M​ inimize false-negative results: ​sample largest ​and ​second largest ​node at each station, especially in adenocarcinoma
• F ​ alse-negative results will occur at a rate of about ​10%​, if only largest lymph node is sampled

EBUS-FNA
➢​Needle:
• ​Size: ​21 and 22 common, ​25
• ​Has multiple ​small dimples o​ n its shaft to enhance echogenicity and improve its screen visualization
​ f penetration
• ​Has variable ​depth o
EBUS-FNA procedure

27/08/2019​2
METHODOLOGY OF ROSE
THE PETHALS AND THORNS OF ROSE Advantages
• ​Reduces the need for additional sampling (including CNB) with a lower risk of procedure complications.
• ​Cost-effective (fewer ancillary techniques).
• ​Decreases the number of passes needed for an adequate sample.
• ​Assists further diagnostic triage (assess whether extra-material is needed, decide how to preserve material for further ancillary
studies).
•​The needle rinses​:
– collected in saline, RPMI, Hanks solution,
formalin, or a preservative solution, like Cytolyt – Processed as a cell block, liquid-based cytology, or
cytospin preparation.
•If ​lung cancer ​: ​additional passes ​for predictive biomarkers.
•If lymphoproliferative disease: material may collected and for ​flow cytometry

Questions to answer at ROSE

• ​Is the specimen representing the target?
– ​Clinical features/ radiology
• ​Is the amount and quality of cells sufficient for a satisfactory morphologic evaluation?
– ​What is the preliminary diagnosis ?
• ​Any ancillary test needed either for diagnosis or for therapy?
– ​How should I handle the specimen ?
ethanol

METHODOLOGY OF ROSE
• ​The material is ​expressed ​over labeled glass slides for ​direct smears​.

• ​Staining by ​rapid stains ​eg, ​Diff-Quik

• ​Evaluated under light ​microscopy ​by the cytopathologist/ cytotechnologist for sample adequacy and a preliminary diagnosis.
formalin

27/08/2019​THE PETHALS AND THORNS OF ROSE Disadvantages

• ​Need of an experienced on-site cytopathologist because relies only on morphology.
• ​Equivocal on-site diagnosis may prematurely end a procedure.
 ​ eed of extra-time from the cytopathologist with financial under compensation of pathologist ́s time.
•N
Cancer Cytopathology 2012

3
27/08/2019​ADEQUACY CRITERIA FOR ROSE
Adequacy for Peribronchial/Peritracheal LNs
• ​There is lack of standardized adequacy criteria
➢​An adequate LN sample:
• ​Lymphocytes ​➢​Adequate: (in general)
➢ ​Inadequate:
• ​Lymphohistiocytic aggregates
​ ave material that explains
• H
• A​ bundant ​bronchial
• ​Germinal center fragments ​the patient’s condition
contamination
• ​Anthracotic pigment-laden macrophages
​ alignant cells
• M
• B​ lood​, ​without ​lymphoid
• G ​ ranulomatous inflammation
cells, granulomatous ​➢​If limited​: qualitative criteria is applied for adequacy •
​ ​Sufficient lymphoid cells
(benign)
inflammation, or malignant cells

4 ​evaluation.
​ e called unsatisfactory
• ​There ​adequacy ​are no universally accepted criteria for EBUS LN ​• ​Any cytological atypia ​should not b

PITFALLS AND CHALLENGES OF INTERPRETATION

• ​Airway elements
• ​Reactive bronchial epithelial cells
• ​Cellular dyscohesion and background necrosis
• ​Signet ring cells
• ​Granulomas
• ​Cytomorphologic overlap
• ​Lymphoproliferative disorders
Reactive
• ​Few cells
• ​Morphologic continuum between benign and abnormal
• ​Cohesive groups
• ​Regular sheets, windows between cells
• ​Knobby contours in groups
• ​No membrane irregularity
• ​Delicate granuler chromatin
– ​Karyopiknosis, karyoreksis,
karyolisis
• ​Cilia/ terminal bar
Carcinoma
To avoid false (+) diagnosis ....
• ​Compare the morphology of atypical cells with

​Do not cells ​trust poor preparation ​

• native
​ ​ ​ ever Know the rely clinical on few context
• • N
cells •​ ​Many cells
• ​Contrast between benign and abnormal
• ​Loss of cohesion
– ​Many single cells
• ​Overlapping, 3-dimention
• ​Smooth contoured groups
• ​Irregular nuclear membrane
• ​Course chromatin
ROLE OF THE CYTOPATHOLOGIST IN LUNG CANCER
• ​Diagnosis
• ​Subtyping
• ​Molecular tests
Applying IHC to small samples ?
• I​ f any doubt in defining squamous or adenoid differentiation, then IHC is needed
• T ​ hreshold for squamous differentiation should be high ​(dense eosnophilic cytoplasm, distinct intercellular borders ?? Not enough!
pseudosquamous adenoCa?)
• ​Use limited IHC to save the tissue for molecular tests , 1 squamous 1 adenoid marker
• ​TTF-1 specific
• ​P40 is more specific than p63
• ​Metastases in lung may be single, large, multiple or lymphangitic.
• I​ n most instances a previous diagnosis of malignancy is available, and confirmation of the clinically suspected lesion is all that is required from the cytopathologist.
• ​However, some pulmonary metastases present before the primary tumour has been identified and may require careful investigation, if inappropriate diagnosis and treatment
of presumed lung primary carcinoma are to be avoided.
• ​Adenocarcinoma, squamous cell carcinoma and small cell carcinoma are histological types that can be difficult to distinguish between primary or secondary tumours.
LUNG CYTOLOGY Primary vs. Metastasis
Squamous
• ​Clusters, streaming, ​whorling

• ​Cell in cell pattern ​• ​Central, oval/ elongated nuclei ​• ​Course dense chromatin

• ​Small nucleoli ​• D​ ense cytoplasm ​• ​Distinct cytoplasmic borders

• K​ eratinized cells
Markers for subtyping
• ​Squamous
P40 P63 CK 5/6 Desmocollin .​..
• ​Adenocarcinoma
TTF-1 Napsin A ​CK 7 ​Mucin stains .​.​nuclear cytoplasmic

.
Adeno
• ​Sheets, papillary ​structures

• ​• • Acini
​ ​Excentric, nuclei round/oval Granular chromatin
Small cell ​
LUNG CARCINOMA ​ morphology
• ​• • Large
​ nucleoli ​Pale/lacy cytoplasm Indistinct borders cytoplasmic •
​ ​Mucin secretion
P63 ​
Yes ​ Positive
No
No

27/08/2019​ Yes

SqCC-small ​cell type

Synaptophysin/ Chromogranin/
​ ​Positive

CD56- ​Yes SCLC

SqCC +++​
P40/CK5
- Or -/+ ​
- TTF1/Napsin A +++
ADC ​
EGFR ​Positive
Yes
Tyrosine ​kinase inhibitors
Crizotinib

ALK ​
Positive No ​Cancer Cytopathology 2011