Transcript Tissue acquisition and reflex testing. How do we prioritize?

Tissue Acquisition and Reflex Testing
How do we Prioritize?
Maureen F. Zakowski, MD
Memorial Sloan-Kettering Cancer Center
November 2013
Testing Principles
• There are no histologic or clinical surrogates
for mutation testing
• All “non squamous” carcinomas of the lung
should undergo mutational analysis
• Giving TKIs to patients without mutations is
harmful (IPASS)
What is “Reflex Testing”?
• Based on pap smear model of testing atypical
specimens for HPV
• No clinical order needed, no discussion, no
requests
• Pathologists sees adenocarcinoma and
specimen is automatically tested for a number
of genetic abnormalities – choice of testing
methods is up to pathologist
Problems in Obtaining Adequate
Specimens
Most lung cancer patients will never come to
surgery and we are left with very limited
samples
The amount of material needed varies with
the testing platform
How Much Material do You Need?
• At least 50% tumor cellularity desirable for Sanger
sequencing; 25% for more sensitive methods such
as Sequenom
• Median DNA yield = 0.76 ug (range 0.16 - 1.12)
• Median tumor cell count = 1373 (range 117 5175)
• Less than 100 cells unsuccessful
Tumor Adequacy
With rare exception all cytology cell blocks
subjectively interpreted as “adequate” for
diagnosis by a pathologist yielded sufficient
quantity and quality of DNA for mutational
analysis
(Advances in Fine Needle Aspiration Cytology for the Diagnosis of Pulmonary
Carcinoma. Hasanovic, Rekhtman, Sigel, Moreira. Pathology Research
International
Volume 2011)
Acquisition
• All IR and EBUS procedures include a cytotech
or fellow on site
• Tissue is analyzed for “adequacy”
• Triage begins here
Prioritization
• Clinical information is essential but often
missing –is this a biopsy to confirm diagnosis
and surgery will follow, or is this all I will ever
get?
• What is the status of the patient? Has the
patient stopped responding to TKIs, is this a
suspected secondary primary?
• Communication is key
Prioritization at MSKCC
• All resected and biopsied adenocarcinomas
are reflexly tested for EGFR/KRAS/ALK
• “non-squamous” is in category
• This is done regardless of stage
• These tumors are is also tested for ALK by IHC
prior to FISH
EML4-ALK Positive Lung Adenocarcinoma
Rearranged ALK
Normal ALK
H&E cell block lung
adenocarcinoma
ALK Ab D5F3
(Cell Signaling)
Abbott-Vysis
ALK FISH assay
ALK IHC now put into clinical use for all adenocarcinomas
Prioritization
• In order to assure adequate material for
mutation testing, great care is needed in
separating adenocarcinoma form squamous to
avoided “wasting” samples needed for
sequencing, etc
• We try to use as few IHC stains as possible
• TTF-1 and p40 are current favorites
Cytology Cell Block
Adenocarcinoma
TTF-1
p40
Cytology Cell Block
Squamous Cell Carcinoma
TTF-1
p40
Assuring Adequacy
• Cytotechs or pathology fellows attend all IR,
EBUS and bronchoscopic procedures when
tissue is biopsied
• Immediate assessment of adequacy is made
• More passes can be requested
• Material is triaged appropriately – lymphoma
for flow etc
‘Keeping Them Honest’
• We do a great deal of QA on our cyto-histo
specimens
• ALK IHC is correlated with FISH results
• Information gathered in reflex testing is used
for many purposes