1. Tissue acquisition and reflex testing. How do we prioritize?
Maureen F. Zakowski, MD
Memorial Sloan-Kettering Cancer Center
November 2013

2. 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)

3. 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

5. 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

6. 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 )
Median tumor cell count = 1373 (range )
Less than 100 cells unsuccessful

7. 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)

8. Acquisition
All IR and EBUS procedures include a cytotech or fellow on site
Tissue is analyzed for “adequacy”
Triage begins here

9. 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

10. 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

11. ALK Ab D5F3 (Cell Signaling)
EML4-ALK positive lung adenocarcinoma
Rearranged ALK
Normal ALK
ALK Ab D5F3 (Cell Signaling)
H&E cell block lung adenocarcinoma
Abbott-Vysis ALK FISH assay V
V5AB
ALK IHC now put into clinical use for all adenocarcinomas 11

12. 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

13. Cytology Cell Block
Adenocarcinoma
TTF-1
p40

14. Cytology Cell Block
Squamous Cell Carcinoma
TTF-1
p40

15. 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

16. ‘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