Increased tumor purity and improved biomarker detection using precision needle punch enrichment of pathology specimen paraffin blocks: Method validation and implementation in a prospective clinical trial.

Abstract

3622 Background: While many sequencing assays may be geared for short variants (SV), more complex biomarkers such as genomic loss of heterozygosity (gLOH) score, also referred to as homologous recombination deficiency (HRD) score, require higher tumor purity for confident detection. Practical methods to increase tumor nuclei percentage (TN%) from pathology specimens are needed to achieve biomarker results to maximize patient matching to approved therapies and/or clinical trial enrollment. Methods: Tumor purity of specimens was determined by the computational analysis pipeline component of the FDA-approved NGS assay, FoundationOneCDx. In the validation study, specimen purities for each tissue block were compared following either no enrichment (UnE, n=46), pathologist-directed enrichment by straight razor blade (RBE, n=30) or precision needle punch (NPE, n=47). Post-enrichment H&E slides confirmed target region sampled for the NPE arm. Based upon validation data, the needle punch process was implemented for the Lung-MAP prospective clinical trial (LM-NPE). TN% was compared between the first 55 tested LM-NPE specimens and the validation study to assess performance on real-world samples outside of a controlled validation experiment. Results: The mean computational TN% in the 4 groups were: UnE: 33%; RBE: 30%; NPE: 52%; and LM-NPE: 48%. In the validation study, NPE had significantly higher purity than both UnE and RBE (p<0.001); in the trial arm, LM-NPE performed equivalently to NPE (p=0.344). Based upon a 30% tumor purity cutoff, gLOH could be determined for 52% UnE, 50% RBE, 89% NPE and 71% LM-NPE. Comparing NPE and LM-NPE groups reveals no statistical difference in Pass/Fail rates for gLOH determination (p=0.883; Fisher’s Test). Conclusions: Precision needle punch cores from tissue blocks have elevated tumor purity, and consequently, a greater number of successful gLOH determinations. Moreover, this process is rapid and inexpensive. Precision punches may constitute best practice with respect to enriching tumor cells from low-purity specimens for biomarker detection in a routine laboratory specimen-processing setting. [Table: see text]