Tissue Acquisition and Reflex Testing How do we Prioritize?

Maureen F. Zakowski, MDMemorial 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 InternationalVolume 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

ALK Ab D5F3 (Cell Signaling)

Normal ALK

Rearranged ALK

Abbott-Vysis ALK FISH assay

H&E cell block lung adenocarcinoma

EML4-ALK Positive Lung Adenocarcinoma

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

Adenocarcinoma

TTF-1 p40

Cytology Cell Block

TTF-1 p40

Squamous Cell Carcinoma

Cytology Cell Block

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