The Use of Direct Smears for Molecular and Immunohistochemical Studies on Cytologic Specimens: A Paradigm Shift for Cytopathologic Technique.
Jeremiah B Placido, Michael H Roh, Bryan L Betz, Lindsay Schmidt, Stewart M Knoepp. University of Michigan, Ann Arbor
Background: As the number of molecular diagnostic and prognostic tests applied to patient specimens continues to grow, it is of paramount importance for cytopathologists to optimize the triage of scarce material obtained from fine needle aspiration biopsies (FNABs). While cell blocks have traditionally served as the mainstay for molecular and immunohistochemical studies performed on FNABs, direct smears serve as a potentially abundant source of material for these ancillary studies.
Design: We first performed a survey of the cellularity of 76 consecutive routine cell blocks prepared from endobronchial ultrasound (EBUS) guided (FNABs) diagnosed as malignant. We next developed and optimized a procedure for fixation and immunocytochemistry on unstained direct smears prepared from FNABs of malignancies. Unstained slides were also optimized for fluorescence in situ hybridization (FISH). Finally, we optimized extraction of DNA for mutational analysis on Diff-Quik stained cytologic smears after verification of tumor cellularity and purity.
Results: Forty-three (57%) of 76 cell blocks from EBUS-guided FNABs of malignancies were either very sparsely cellular or acellular. Next, immunocytochemistry was performed on 52 unstained direct smears from malignant cytologic specimens utilizing the Ventana autostainer after brief formalin fixation and antigen retrieval. The cases included pulmonary squamous cell carcinoma (n=15), pulmonary adenocarcinoma (n=27), metastatic melanoma (n=6), and Hodgkin lymphoma (n=4); antibodies directed against TTF-1, Napsin A, p63, S-100, Mart1, Melan-A, HMB-45, CD15, and CD30 were utilized. Furthermore, DNA extraction from Diff-Quik stained smears of pulmonary adenocarcinoma for EGFR and KRAS mutation was successful in all performed cases (n=33). Activating mutations for EGFR were found in 3/33 cases, and KRAS was mutated in 11/33 cases. Finally, FISH for EWS rearrangement utilizing unstained direct smears was performed on two cases of Ewing sarcoma.
Conclusions: The immediate triaging of cellular material during the time of FNAB for successful immunocytochemical and molecular studies represents a major advance in diagnostic cytopathology. The ability to guarantee that sufficient material is present for ancillary studies, during on-site assessment, represents a paradigm shift for cytopathologic workup. It is anticipated that these methods will become the standard of care as increasing demands are placed on cytopathologists to render additional molecular diagnostic and prognostic information on routine cytology specimens.
Monday, February 28, 2011 8:00 AM
Platform Session: Section G 1, Monday Morning