High-Throughput Molecular Analysis from Leftover of Fine Needle Aspiration Cytology of Mammographically Detected Breast Cancer

Abstract

We investigated whether residual material from diagnostic smears of fine needle aspirations (FNAs) of mammographically detected breast lesions can be successfully used to extract RNA for reliable gene expression analysis. Twenty-eight patients underwent FNA of breast lesions under ultrasonographic guidance. After smearing slides for cytology, residual cells were rinsed with TRIzol to recover RNA. RNA yield ranged from 0.78 to 88.40 µg per sample. FNA leftovers from 23 nonpalpable breast cancers were selected for gene expression profiling using oligonucleotide microarrays. Clusters generated by global expression profiles partitioned samples in well-distinguished subgroups that overlapped with clusters obtained using "biologic scores" (cytohistologic variables) and differed from clusters based on "technical scores" (RNA/complementary RNA/microarray quality). Microarray profiling used to measure the grade of differentiation and estrogen receptor and ERBB2/HER2 status reflected the results obtained by histology and immunohistochemistry. Given that proliferative status in the FNA material is not always assessable, we designed and performed on FNA leftover a multiprobe genomic signature for proliferation genes that strongly correlated with the Ki67 index examined on histologic material. These findings show that cells residual to cytologic smears of FNA are suitable for obtaining high-quality RNA for high-throughput analysis even when taken from small nonpalpable breast lesions.

Liquid-Based Cytology of the Breast: Pitfalls Unrecognized before Specific Liquid-Based Cytology Training - Proposal for a Modification of the Diagnostic Criteria

“…It also permits the preparation of additional samples [24], facilitating the application of special techniques such as immunohistochemistry and molecular biology [25,26]. It is thus likely that pathologists will be increasingly exposed to LBC in evaluating the breast and other organs, including metastatic sites.…”

Reverse transcription–polymerase chain reaction molecular testing of cytology specimens: Pre‐analytic and analytic factors

“…Prompt cooling of nonfixed tissue specimens on wet ice after devitalization slows the cellular metabolism and is recommended to minimize RNA degradation . With respect to the processing of fresh cytologic material, high‐quality RNA has been obtained from studies performing dedicated FNA for routine cytologic diagnosis and dedicated single or multiple FNAs for RNA extraction as well as from cells residual to diagnostic cytologic smears of FNAs . For example, after smearing slides from FNA specimens of small, nonpalpable breast lesions for cytologic examination, Annaratone et al rinsed residual cells in TRIzol and recovered a mean RNA yield per FNA of 11.7 μg with confirmation of RT‐PCR efficiency with a housekeeping gene ( PGK ) in 92.8% of the samples analyzed.…”

Lymph Node Fine-Needle Cytology: Beyond Flow Cytometry

“…The incorporation of GEP array information requires the extraction of good-quality DNA and RNA from tissues or blood, and often the need for fresh samples is a logistic challenge that is difficult to overcome in routine practice. Therefore, not surprisingly, FNC samples have been conveniently used for the GEP of different tumors [63,64], including lymphomas [65], exploiting the specific advantages of FNC - namely to harvest vital tumor cells avoiding stroma, fat and other nontumor components. GEP improves the current prognostic indexes based on clinical criteria, such as the International Prognostic Index (IPI) [60,66], because the corresponding GEP-based prognostic models are different in each pathological entity.…”