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Pulmonary & Mediastinal Small Biopsy-Cytology: Case Studies with Current Guidelines & Trends in Specimen Management
Andre Moreira, MD, PhD Memorial Sloan Kettering Cancer Center
Anjali Saqi, MD, MBA Columbia University Medical Center
CONFLICT OF INTEREST
In the past 12 months, we have not had a significant financial interest or other relationship with the manufacturer(s) of the product(s) or provider(s) of the service(s) that will be discussed in my presentation.
CASE 1
CASE 1
• 79-year-old man with hemopytsis
• Chest CT-scan: 2.9 cm lung mass and enlarged lymph
node
• Endobronchial ultrasound (EBUS)-guided fine needle
aspiration with rapid on-site evaluation (ROSE)
• Based on the following images, what should be
communicated to the pulmonologist performing the
procedure?
Based on the images, what should be communicated
to the pulmonologist performing the procedure?
A. Lymphocytes and carcinoma; stop procedure.
B. Lymphocytes and histiocytes; continue procedure.
C. Lymphocytes and bronchial cells; stop procedure.
D. Lymphocytes and granuloma; continue procedure.
ADEQUACY:
EBUS LYMPH NODES
• Identification of one of the following:
– Neoplasm (e.g., carcinoma, lymphoma)
– Granuloma*
• Potential pitfalls
– False positive
• Misinterpretation of incidental/reactive bronchial cells or
pneumocytes
– False negative
• Scant malignant cells
• Malignant cells obscured by bronchial cells
ADEQUACY: EBUS LYMPH NODES
• Adequacy of lymph node
without neoplasm or granuloma
• Important to avoid false
negative diagnosis
• Criteria?
– No strict guidelines
– No “minimum” passes
– Count lymphocytes and/or
(pigmented) macrophages*
– ↑ lymphocytes = ↓ false
negative
Karunamurthy
et al.
Unsatisfactory Less than optimal Satisfactory Interpretation
Number of lymphocytes/high power field (HPF)
0 <40/HPF >40/HPF Higher false
negativity in
unsatisfactory and
less than optimal
cases.
Fibrosis/hylanization
due to Hodgkin
lymphoma or
treatment related
changes have lower
cellular yield.
Alsharif M et al 0 1 3
Number of lymphocytes
<40 41-200 >200 Confluent sheets or germinal centers True negatives have
scores of 2 or 3
Nayak A et al Inadequate Adequate* Adequate*
Number of lymphocytes/low power field (LFP) (x100)
Either “Adequate”
category is not
satisfied.
Germinal Center fragments >5 fields with >
100 /LPF and <2
groups
of bronchial
cells/LPF
If not adequate, re-
aspirate lymph node
or aspirate another
lymph node
Crapanzano JP, Saqi A. Adequacy and Tissue Preservation of Small Biopsy and Cytology Specimens. Diagnosing Non-Small Cell Carcinoma in Small Biopsy and Cytology. Springer, 2014.
1. Lymphocytes and histiocytes present; stop
procedure.
2. Lymphocytes and histiocytes present; continue
procedure.
3. Bronchial cells present; stop procedure.
4. Carcinoma present; stop procedure.
Key point(s): • Ensure that there are adequate numbers of lymphocytes to avoid a false
negative diagnosis and increase the negative predictive value.
CASE 2
CASE 2
• 72-year-old male with 6.1 cm lung mass
• CT-guided FNA was performed
• What is the best diagnosis based on the following images?
What is the best diagnosis based on the following images?
A. Adenocarcinoma; submit for molecular studies.
B. Squamous cell carcinoma; do not submit for molecular studies.
C. Adenosquamous carcinoma; submit for molecular studies.
D. Mucoepidermoid carcinoma; do not submit for molecular studies.
EVOLUTION OF CLASSIFICATION:
SINGLE-STEP MULTI-STEP
Small cell carcinoma NSCLC
Histological (NSCLC)
Driver mutations (ADCA)
Adapted from Kris MG. Pao W, Girard N. Lancet Oncol 2011. Harris T. Discovery Medicine, 2012.
Adenocarcinoma
Squamous cellcarcinoma
Large cellcarcinoma
Traditional
CLASSIFICATION FOR SMALL BIOPSY/CYTOLOGY (IASLC/ATS/ERS)
Adopted from: Travis WD, Rekhtman N. 2011.
Avoid “inadequate” positives “Neoplasm” or “malignancy” is inadequate Minimize “NSCLC-NOS” diagnosis
Solution: Doing More With Less
Diagnosis: Carcinoma
Morphology
Immunostains
Moleculardiagnosis
Triage
Morphology
Technicalquality
Procure & Triage
Prepare Slides
Evaluate
Expel specimen on
slide
Identify & select tissue
Prepare 2 smears
Air dry & stain 1 smear
Perform ROSE
Diagnosis?
AS?
Yes
Terminate procedure
No
Repeat procedure
Alcohol fix 1 smear
Rinse needle Allocate
remaining for AS
Solution: Develop Algorithm
Smear Preparation
OPTIMAL SMEARS:
PICK AND SMEAR
Suboptimal Triage & Smear Preparation Optimal Triage & Smear Preparation
CELL BLOCKS
• Advantages
– Architectural detail
– Preservation of original smears
– Future diagnostic studies
• Current state of cell blocks
– Variable cellular yield1,2
– >10 methods to prepare cell blocks
0 10 20 30 40 50 60
Yes
No
Sometimes
N/A
Satisfaction With Cell Block Quality
1Yung, Otell et al. 2012. 2Billah et al
TTF-1 (+) CK5 (+) Also p63 (+)
p63
TTF-1
ADENOSQUAMOUS CARCINOMA
Rekhtman N. Clin Cancer Res, 2012.
Adenocarcinoma Adenosquamous carcinoma
Squamous cell carcinoma
EGFR Yes Yes* No
• Adenosquamous carcinoma represents 0.4 to 4% of NSCLC • Similar rate as adenocarcinoma • Mutations present uniformly in glandular and squamous components • IASLC, ATS, ERS: Submit for testing if a component of adenocarcinoma cannot
entirely be excluded.
GLANDULAR AND SQUAMOUS COMPONENTS
PAX-8
METASTATIC ENDOMETRIOID CARCINOMA
What is the best way to manage this specimen based on the following images?
1. Adenocarcinoma; submit for molecular studies.
2. Squamous cell carcinoma; do not submit for molecular studies.
3. Adenosquamous carcinoma; submit for molecular studies.
4. Mucoepidermoid carcinoma; do not submit for molecular studies
Key point(s): • Minimize use of non-small cell carcinoma, NOS diagnosis. • Develop algorithm to triage specimen for ancillary studies. • Submit small biopsy or cytology samples for molecular testing when there
is any adenocarcinoma component or an adenocarcinoma component cannot entirely be excluded.
CASE 3
CASE 3
• 66-year-old woman with 3 cm peripherally-located lung
mass
• FNA with rapid on-site evaluation (ROSE)
• CT scan shows needle in lesion
• Per radiologist: mass is firm
• Three FNA passes show rare cells and mostly blood
• Based on the representative image of the FNA, what
should be communicated to the radiologist?
Based on the representative image from the FNA, what should be communicated to the radiologist?
A. Recommend additional CT-guided FNA.
B. Recommend endobronchial ultrasound (EBUS)-guided FNA.
C. Recommend core biopsy.
D. Recommend surgery.
Based on the representative image of the FNA, what should be communicated to the radiologist?
A. Recommend additional CT-guided FNA
B. Recommend endobronchial ultrasound (EBUS)-guided
C. Recommend core biopsy
D. Recommend surgery
Key point(s): • In cases with scant cellularity, consider requesting a core biopsy.
• Touch preparation with rapid on-site evaluation (ROSE)
performed
• Based on the images, what is the best diagnosis?
Based on the images, what is the best diagnosis?
A. Benign: Organizing pneumonia
B. Benign: Granuloma
C. Malignant: Sarcomatoid carcinoma
D. Malignant: Leiomyosarcoma
FNA VS
CORE BIOPSY
• No consensus for modality of choice
• Influenced by several variables
– Preference/comfort of radiologist and pathologist
– Availability of ROSE
– Risk to patient
– Nature of lesion
• Equivalent at classifying NSCLC
Coley SM, Crapanzano JP, Saqi A. Cancer Cytopatholgy 2015;.
0
5
10
15
20
25
30
35
40
45
50
FNA Core Both
Nu
mb
er o
f C
ase
s
Sampling Modality
Total
SpecificDiagnosis
0
5
10
15
20
25
FNA Core Both
Nu
mb
er
of
Cas
es
Sampling Modality
Total cases
Molecular work-upperformed
Specific Diagnosis in Cases of Carcinoma* Primary Lung Adenocarcinomas with Molecular Work-up*
Touch Preparations
Advantages
• High diagnostic accuracy for malignancy
• Provide preliminary diagnosis
• Adequate sampling
• Appropriate triage
Disadvantages
• Transfer of diagnostic cells onto touch prep
• Sample single area with single pass (FNA samples wider area)
• Diagnostic clues different from smears*
• Drying artifact for alcohol fixation*
• Crush artifact*
Complication rate?
FNA vs Core Biopsy
Scant cellularity
Organizing pneumonia
Spindle cell neoplasm
Nodular sclerosis
Hodgkin’s lymphoma
Saqi A, Coley SM, Crapanzano JP. Cytojournal. 2014 Jan
TOUCH PREPS TECHNIQUES
Suboptimal Optimal
Limit single section per slide
More slides are available for ancillary testing
Minimize tissue loss
Avoid excessive trimming of paraffin block
ROSE
• Order blank slides for ancillary studies upfront
No ROSE
•Cut blank upfront and/or save intervening levels
•At time of IHC order, request blanks
Create >1 block when possible Core biopsies
Divide cores/fragments into multiple blocks
Dedicated passes
Place cores directly into formalin (RPMI etc.)
Saqi A, Crapanzano JP. Adequacy and Tissue Preservation of Small Biopsy and Cytology Specimens. Diagnosing Non-Small Cell Carcinoma in Small Biopsy and Cytology. Moreira AL and Saqi A, Editors. Springer, 2014.
Organizing Pneumonia
Organizing Pneumonia
Granuloma
Granuloma
Sarcomatoid Carcinoma
Metastatic Leiomyosarcoma
Mesenchymal tumors
Primary
Spindle cell carcinoid
Thymoma
Solitary fibrous tumor
Neurogenic tumor
Sarcomatoid carcinoma
Synovial sarcoma
Other
Secondary
Spindle cell melanoma
Benign metastasizing
leiomyoma
Sarcomatoid mesothelioma
Metastatic sarcoma
Synovial sarcoma
Differential For Mesenchymal Tumors of the Lung And Mediastinum
Monaco SE, Dacic S. Adequacy and Tissue Preservation of Small Biopsy and Cytology Specimens. Diagnosing Non-Small Cell Carcinoma in Small Biopsy and Cytology. Moreira AL and Saqi A, Editors. Springer, 2014.
IHC: Neoplasms with Spindle Cells
Keratin(s) TTF-1 CD34 BCL2 Desmin SMA ER PR
Sarcomatoid carcinoma + +/-
Mesothelioma +
Synovial sarcoma +
Solitary fibrous tumor + +
Benign metastasizing
leiomyoma
+ + + +
Leiomyosarcoma + +
Monaco SE, Dacic S. Adequacy and Tissue Preservation of Small Biopsy and Cytology Specimens. Diagnosing Non-Small Cell Carcinoma in Small Biopsy and Cytology. Moreira AL and Saqi A, Editors. Springer, 2014.
Angiosarcoma
CK CK7
Based on the images, what is the best diagnosis?
A. Benign: Organizing pneumonia
B. Benign: Granuloma
C. Malignant: Sarcomatoid carcinoma
D. Malignant: Leiomyosarcoma
Key point(s): • Scant cellularity is typically seen in cases of spindle cell lesions, Hodgkin
lymphoma, and organizing pneumonia. • Spindle cells can be seen in benign and malignant entities. • Neoplasms with spindle cells may represent primary or metastatic
neoplasms. • IHC is typically necessary to classify the malignancy. • Triage to ensure sufficient sample for ancillary studies.
CASE 4
CASE 4
• 79-year-old man, former smoker, with 5 cm lung mass
and lymph node metastases
• Poor surgical candidate
• Treated with chemotherapy but disease progressed
• Possible candidate for immunotherapy
• Based on the clinical history, what additional step(s)
should be taken?
p40
• Based on the clinical history, what additional step(s)
should be taken?
A. Submit sample for molecular studies.
B. Perform CD45 immunohistochemical stain.
C. Perform CD163 immunohistochemical stain.
D. Perform programmed death ligand-1 (PD-L1)
immunohistochemical stain.
Immunotherapy blocks PD-1
pathway to help prevent cancer
cells from hiding
https://www.keytruda.com/non-small-cell-lung-cancer/how-keytruda-works/;
IMMUNOTHERAPY • Programmed death receptor-1
(PD-1) is expressed by T-cells
• Programmed death ligand-1 (PD-L1) is the ligand for PD-1
• Binding of PD-1 to PD-L1 on tumor cell can diminish the function of the T-cells
• Drugs developed to disrupt this interaction
• Antibodies target PD-1
• Second-line therapies
– Progression following platinum chemotherapy or EGFR/ALK-targeted therapy
https://pbs.twimg.com/media/CmMCAJzUsAACFDM.jpg
FDA-approved Immunotherapies for NSCLC
Pembrolizumab (Keytruda)
• NSCLC
• Companion (FDA-approved) immunohistochemical test – Required for safe & effective
use of drug
– > 50% staining (Dako Pharm Dx 22C3)
• Survival benefit with staining > 50% staining
Nivolumab (Opdivo)
• NSCLC
• Complementary immunohistochemical test – Not essential for determining
who should receive drug
– > 1% staining (Dako 28-8 Pharm Dx)
• ?
PEMBROLIZUMAB VERSUS DOCETAXEL FOR
PREVIOUSLY TREATED NSCLC
KEYNOTE-010 randomized controlled trial for advanced NSCLC. Kaplan-Meier analysis of overall survival. (A) For patients with a PD-L1
tumor proportion score of >50%.
(B) For all patients
Herbst RS, Baas P et al. Pembrolizumab versus docetaxel for previously treated, PD-L1-positive, advanced non-small-cell lung cancer (KEYNOTE-010): a randomised controlled trial. Lancet. 2016 Apr 9;387(10027):1540-50.
GARON EB ET AL. N ENGL J MED 2015;372:2018-2028.
PD-L1 Expression in Non–Small-Cell Lung Cancers.
Figure 1. PD-L1 Expression in Non–Small-Cell Lung Cancers. Results were reported as the percentage of neoplastic cells showing membranous staining of programmed cell death ligand 1 (PD-L1) (proportion score). Shown are tumor samples obtained from patients with a proportion score of less than 1% (Panel A), a score of 1 to 49% (Panel B), and a score of at least 50% (Panel C) (all at low magnification). Tumor samples with the corresponding proportion scores are shown at a higher magnification in Panels D through F.
NIVOLUMAB VERSUS DOCETAXEL IN ADVANCED
SQUAMOUS-CELL NON–SMALL-CELL LUNG CANCER
“Among patients with advanced, previously-treated squamous –cell NSCLC, overall survival, response rate, and progression-free survival were significantly better with nivolumab than with docetaxel, regardless of PD-L1 expression level.
Brahmer J, Reckamp KL et al. N Engl J Med. 2015 Jul 9; 373(2): 123–135.
PD-L1 SCORING CRITERIA
• Multiple monoclonal antibodies against PD-1 or PD-L1 tested in pulmonary carcinomas.
• Each therapeutic antibody with corresponding PD-L1 immunohistochemistry.
• Different antibodies detection system,s and scoring-criteria.
Scheel AH et al. Harmonized PD-L1 immunohistochemistry for pulmonary squamous-cell and adenocarcinomas. Mod Pathol. 2016 Jul 8.
• Immunotherapies and parallel development of diagnostic tests (companion and complementary)
• Lack of common biomarker/harmonization
• One test cannot be applied to another agent
Problem Potential Solution?
• Standardization
• Blueprint proposal FDA/ASCO/AACR
– Four pharmaceutical companies
– Two diagnostic companies
• Assess similarities and differences among assays
Hansen AR, Siu LL. JAMA Oncol. 2016;2(1):15-16. doi:10.1001/jamaoncol.2015.4685
• Based on the clinical history, what additional step(s) should be taken?
A. Submit sample for molecular studies.
B. Perform CD45 immunohistochemical stain.
C. Perform CD163 immunohistochemical stain.
D. Perform programmed death ligand-1 (PD-L1) immunohistochemical stain.
• Based on the clinical history, what additional step(s) should be taken?
A. Submit sample for molecular studies.
B. Perform CD45 immunohistochemical stain.
C. Perform CD163 immunohistochemical stain.
D. Perform programmed death ligand-1 (PD-L1) immunohistochemical stain.
Key point(s): • Companion diagnostics are required whereas complementary are not. • Only two immunotherapies are currently FDA-approved. • Each therapy, including those in clinical trials, have different
immunohistochemical antibodies. • In most, only the tumor cells are evaluated.
Questions?