Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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The identification of challenges in tissue collection for biomarker studies: the Q-CROC-03 neoadjuvant breast cancer translational trial experience.

Author(s): Aguilar-Mahecha A, Lafleur J, Pelmus M, Seguin C, Lan C, Discepola F, Kovacina B, Christodoulopoulos R, Salvucci O, Mihalcioiu C, Roy JA, Robidoux A, Marcus EA, Batist G, Basik M

Publication: Mod Pathol, 2017, Vol. , Page

PubMed ID: 28752846 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared morphology, DNA and RNA yield, and RIN among tumor specimens preserved by snap-freezing, RNAlater, and formalin fixation paraffin embedding. Specimens used in the study included core needle biopsy and residual tumor specimens from 60 triple negative breast cancer patients. Three to four core needle biopsies were obtained with a 14- or 16-gauge needle prior to chemotherapy (n=207) and again prior to surgery (3-4 weeks after completion of 4-6 months of neoadjuvant treatment, n=91). When available, three to four fragments of residual tumor were taken from the surgical specimen (n=105). Specimens at each collection timepoint were divided and aliquots were snap-frozen, placed in RNAlater and immediately frozen, or placed in 10% formalin. Frozen specimens were stored at -80°C and shipped in batches on dry ice to the central laboratory where they were embedded in OCT. Formalin specimens were immediately shipped at 4°C to the central laboratory and embedded in paraffin within 48 hours of collection. Sections were stained with H&E; examined by a pathologist for tumor content, stroma, and necrosis; and in cases of low tumor content, enriched by macrodissection for nucleic acid extraction. DNA and RNA were extracted from 93 specimens (71 snap-frozen and 21 RNAlater) that met the cellularity threshold (≥50%) with the AllPrep DNA/RNA/miRNA Universal Kit. DNA yield, RNA yield, and 260/280 ratios were measured by spectrophotometry. RNA integrity as determined by RIN was performed using a bioanalyzer.

   Summary of Findings:

   Poor preservation of nuclear morphology was observed in 45% of RNAlater specimens, rendering pathological evaluation challenging. In contrast, moderate or good morphology was observed in 75% of snap-frozen specimens and >90% of FFPE specimens. Although not statistically significant, twice as many RNAlater specimens were not evaluable compared to snap-frozen specimens and four times as many RNAlater specimens were not evaluable compared to FFPE specimens. The percentage of specimens that met the ≥50% cellularity threshold for molecular analyses was similar for the three preservation methods (P=0.64). Residual tumor specimens preserved in RNAlater yielded significantly less DNA compared to snap-frozen specimens (P<0.001). While not statistically significant, average RNA yield was lower in RNAlater-preserved specimens compared to snap-frozen specimens (4.92 μg versus 17.5 μg in biopsies, P=0.22; 17.2 μg versus 56.52 μg in surgical tissues, P=0.09). Average RIN values were 7.6 (range 3.4–9.1) for snap-frozen and 6.2 (range 1.8–8.7) for RNAlater specimens. The authors noted that   RNA quality was not affected in specimens that were macrodissected.

   Biospecimens

   • Tissue - Breast

   Preservative Types

   • Formalin
   • RNAlater
   • Frozen

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   Morphology	H-and-E microscopy
   RNA	Automated electrophoresis/Bioanalyzer
   RNA	Spectrophotometry
   DNA	Spectrophotometry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	Formalin (unbuffered) Snap frozen RNAlater
   Biospecimen Acquisition	Method of tissue acquisition	Core needle biopsy Surgical resection

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