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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?