Molecular Detection of Metastatic Cancer in Cell-Free Cytocentrifugation Supernatant Fluid from Needle Aspirates of Lymph Nodes
Allan R Smith, Jan F Silverman, Yulin Liu, Uma Krishnamurti, Shahid Bokhari, Candy Binkert, Beth Ujevich, Alok Mahonty, Sidney D Finklestein. Allegheny General Hospital, Pittsburgh, PA; RedPath Integrated Pathology, Inc., Pittsburgh, PA
Background: Cytologic diagnosis of lymph node needle aspirates for the presence or exclusion of metastatic cancer can be difficult leading to occasional indeterminate diagnosis especially if only a few atypical cells are present. We explored whether cell free DNA from the lymph node aspirate could serve as an independent source for mutational analysis with the goal to improve the detection or exclusion of malignancy.
Design: 8 needle aspirate of lymph nodes with metastatic cancer (n=4) and without cancer (n=4) were used in this pilot study. In each case, DNA was extracted from 2 ml of cell-free cytocentrifugation supernatant fluid from residual aspirate not utilized in preparing direct smears. DNA quantity measured by optical density. DNA amplifiability was assessed using qPCR. Mutational analysis followed using PCR/capillary electrophoresis for a broad panel of markers (KRAS point mutation by sequencing, microsatellite fragment analysis for loss of heterogeneity [LOH] of 16 markers at 1p, 3p, 5q, 9p, 10q, 17p, 17q, 21q, 22q). In selected cases, microdissection of stained cytology smears and/or cytocentrifugation cellular slides were similarly analyzed and compared.
Results: No detectable mutations were present in cytologic negative cases. All metastatic carcinoma cases showed extensive detectable mutations in both the microdissected tumor and corresponding cytocentrifugation supernatant fluid. The mutational profile between the malignant cells and supernatant was highly concordant including involvement of specific parental alleles when LOH was present. The supernatant fluid of all 4 cancer specimens showed additional mutations not present in the microdissected tumor. Also, for every mutation present in both specimen, the clonality of mutational change was equal to or higher in the supernatant fluid sample compared to the microdissected tumor cells.
Conclusions: 1) The cytocentrifugation supernatant fluid, gathered during cytology preparation, and typically discarded, contains adequate levels of analyzable DNA suitable for mutation detection and characterization. 2) The greater content and higher clonality of mutational change in the supernatant fluid of lymph node aspirates affords a simple way to improve the detection or exclusion of cancer in cases with a limited number of atypical cells. 3) Molecular analysis of supernatnat fluid can be especially helpful for the detection of malignancy. The results support further studies to evaluate its clinical utility.
Wednesday, March 21, 2012 9:30 AM
Poster Session V # 263, Wednesday Morning