Resource-efficient pooled sequencing expands translational impact in solid tumors.

Renzo Dinatale,Memorial Sloan Kettering Cancer Center, New York, Usa ,Vladimir Makarov,James Hsieh,Roy Mano,Angela Yoo,Ed Reznik,Benjamin Freeman,Nicole Rusk,Paul Russo,Andrew Winer,Michael Berger,Ari Hakimi,Timothy Chan,Ying-Bei Chen,Esther Drill,Jonathan Coleman,Irina Ostrovnaya,Alexander Sankin

doi:10.52733/kcj18n2.a1

Abstract

Intratumoral genetic heterogeneity (ITH) poses a significant challenge to utilizing sequencing for decision making in the management of cancer. Although sequencing of multiple tumor regions can address the pitfalls of ITH, it does so at a significant increase in cost and resource utilization. We propose a pooled multiregional sequencing strategy, whereby DNA aliquots from multiple tumor regions are mixed prior to sequencing, as a cost-effective strategy to boost translational value by addressing ITH while preserving valuable residual tissue for secondary analysis. Focusing on kidney cancer, we demonstrate that DNA pooling from as few as two regions significantly increases mutation detection while reducing clonality misattribution. This leads to an increased fraction of patients identified with therapeutically actionable mutations, improved patient risk stratification, and improved inference of evolutionary trajectories with an accuracy comparable to bona fide multiregional sequencing. The same approach applied to non-small-cell lung cancer data substantially improves tumor mutational burden (TMB) detection. Our findings demonstrate that pooled DNA sequencing strategies are a cost-effective alternative to address intrinsic genetic heterogeneity in clinical settings.

Highlights

Intratumoral genetic heterogeneity (ITH) poses a significant challenge to utilizing sequencing for decision making in the management of cancer
We demonstrate that DNA pooling from as few as two regions significantly increases mutation detection while reducing clonality misattribution
As a result of ITH, clinically informative but subclonal mutations are commonly missed by the standard practice of sequencing single tumor regions

Summary

Translational Impact in Solid Tumors

Ari Hakimi * b,c a Computational Oncology Service, Epidemiology & Biostatistics Department, Memorial Sloan Kettering Cancer Center, New York, USA. C Urology Service, Department of Surgery, Memorial Sloan Kettering Cancer Center, New York, USA. D Biostatistics, Epidemiology & Biostatistics Department, Memorial Sloan Kettering Cancer Center, New York, USA. H Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, USA. We demonstrate that DNA pooling from as few as two regions significantly increases mutation detection while reducing clonality misattribution. This leads to an increased fraction of patients identified with therapeutically actionable mutations, improved patient risk stratification, and improved inference of evolutionary trajectories with an accuracy comparable to bona fide multiregional sequencing. Complete loss of spatial information could potentially be avoided (with an increase in cost and overhead) by barcoding DNA libraries before sequencing, an approach that has previously been demonstrated by several

Clonality misattribution

Findings

Sequencing Conventional