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Hormone receptor and HER2 assessment in breast carcinoma metastatic to bone: A comparison between FNA cell blocks and decalcified core needle biopsies.

Author(s): Zeng J, Piscuoglio S, Aggarwal G, Magda J, Friedlander MA, Murray M, Akram M, Reis-Filho JS, Weigelt B, Edelweiss M

Publication: Cancer Cytopathol, 2020, Vol. 128, Page 133-145

PubMed ID: 31883437 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared FFPE FNA specimens to matched decalcified FFPE CNB specimens for ER, PR, and HER2 IHC analysis in bone metastases of breast cancer patients. Concordance between the ER, PR, and HER2 status of the metastatic bone biopsy and primary breast tumor was also investigated. Cases with discordant ER IHC staining results were further analyzed by real-time qRT-PCR quantification of ESR1 expression levels. Archived FFPE FNA and CNB tumor specimens from 27 female patients (36-81 y, mean age 55) with breast carcinoma metastatic to bone with prior ER, PR, and HER2 IHC results (n=27) and HER2 fluorescence in situ hybridization (FISH) results (n=22) for the primary breast cancer were included in the study. Five patients had a bone metastasis diagnosed at the time of primary diagnosis while 22 patients had a bone metastasis-free interval of 5-12 y. Archived FNA cell blocks had been fixed in 10% neutral buffered formalin in an automated tissue processor for 6 hours followed by paraffin embedding, cutting, and staining. Archived bone CNB specimens had been fixed in 10% neutral buffered formalin for 6-7 h; rinsed in water for 15–30 min; placed in a fixative/decalcifier solution containing formic acid, formaldehyde, EDTA, and deionized water for 1 h; placed a tissue processor cassette; and then processed by a standard 2.5 h protocol (details not provided). Five μm-thick sections of each matched pair of specimens were stained for IHC analysis of ER, PR, and HER2 and assessed by two independent pathologists. The proportion of positive tumor cells were divided into six categories: 0 (negative), 1 (<1%), 2 (1–10%), 3 (11–33%), 4 (34–66%), and 5 (67–100%). Staining intensity was scored as 0 (no staining), 1 (weak), 2 (moderate), or 3 (strong). ER and PR scoring was calculated by adding the proportion and intensity of positively stained tumor cells for a total score ranging from 0-8. Cases were classified as negative if <1% of the tumor cells labeled for the hormonal receptors or had a staining score from 0-2. HER2 staining was classified as negative (0 or 1+), equivocal (2+), or positive (3+). For cases with discordant FNA and CNB ER IHC staining results (n=18), RNA was extracted from 8 μm-thick sections using the RNeasy FFPE RNA Isolation Kit and quantified using a Qubit Fluorometer. ESR1 mRNA  levels were quantified by real-time qRT-PCR and normalized using two reference genes (TFRC and MRPL19).

   Summary of Findings:

   Concordance for ER expression between FFPE FNA and decalcified FFPE CNB specimens was 89% (24/27), with 11% (3/27) of cases classified as ER-negative in both the FNA and decalcified CNB specimens (100% concordance) and 89% (24/27) FNA specimens and 78% (21/27) decalcified CNB specimens classified as ER-positive (88% concordance). In the three discordant cases, ER staining in the FFPE FNA specimen ranged from 5–90% but was completely absent in the matched FFPE decalcified CNB specimen. Estrogen receptor alpha (ESR1) mRNA levels were comparable in most cases and only differed between the FFPE FNA and decalcified CNB specimen in one of the thirteen cases examined. Concordance in ER IHC expression between primary breast tumors and matched bone metastases specimens was 81% (22/27 cases) for FNA specimens and 78% (21/27) for decalcified CNB specimens. Overall concordance for PR staining between FFPE FNA and decalcified CNB specimens was 67% (18/27 cases). Of the nine discordant cases, six decalcified CNB specimens exhibited no staining or a decrease >30% in tumor cell staining compared to the matched FNA specimen while three cases were PR-positive in the decalcified CNB specimen but PR-negative in the FNA specimen. PR IHC concordance rates were similar between primary breast tumors and bone metastases sampled by FNA and by decalcified CNB (48% and 44%, respectively). Concordance of HER2 IHC staining of FFPE FNA and decalcified CNB specimens was 93% (25/27), with 21 cases classified as negative (0–1+) in both specimens, two cases in which both specimens were classified as equivocal (2+), and two cases classified as positive in both specimen types (3+). In both of the discordant cases, the FNA specimen was classified as equivocal and the matched decalcified CNB specimen was negative, although both cases had low HER2 amplification during prior FISH analysis of the primary tumor. Concordance of HER2 status between the primary tumor and both metastatic specimens (CNB and FNA) was 86% (19/22 cases).

   Biospecimens

   • Tissue - Bone
   • Tissue - Breast

   Preservative Types

   • Formalin

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   RNA	Fluorometry
   Protein	Immunohistochemistry
   DNA	FISH
   RNA	Real-time qRT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Method of tissue acquisition	Core needle biopsy Fine needle aspiration
   Biospecimen Acquisition	Biospecimen location	Primary breast tumor Breast cancer metastases to bone
   Immunohistochemistry Specific	Targeted peptide/protein	HER2 ER PR
   FISH Specific	Targeted nucleic acid	HER2
   Real-time qRT-PCR Specific	Targeted nucleic acid	ESR1

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