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In instances where tissue biopsy is not medically prudent or tumor tissue material is insufficient for molecular testing, alternative methods are needed. Since cell-free DNA (cfDNA) has been shown to provide a representative surrogate for tumor tissue, we sought to evaluate its utility in this scenario. Here we present data from 91 patients with known neoplasms whose tumors were not able to be tested using conventional tissue biopsy. The cfDNA from each patient was assayed with low-coverage (~0.3X) genome-wide sequencing and copy number alteration (CNA) events were identified and characterized using analytical methods originally developed for noninvasive prenatal testing (NIPT). To quantify the level of CNAs present in the plasma of cancer patients, we utilized the genomic instability number (GIN). First, the technical variability of the GIN was evaluated in a large sample cohort. We therefore processed genome-wide sequencing results from 27,742 pregnant women who consented to have their samples used for research and whose NIPT results yielded no detected CNAs. Utilizing these data, we established a threshold whereby we could differentiate technical noise from biological signal when CNAs were present down to a 1% ctDNA fraction with 99.7% specificity. Applied broadly, CNAs were detected in 32 of the 91 patients with difficult-to-biopsy cancers evaluated (35%) with detection frequencies tightly linked to cancer type. Importantly, there was a significant reduction in progression-free survival for the first treatment after liquid biopsy for patients with an elevated GIN relative to those without, suggesting a prognostic link between GIN and patient outcome across a wide variety of tumor types. In addition, samples with an elevated GIN also showed a significant concomitant reduction in cfDNA fragment length, consistent with previous reports suggesting that cfDNA derived from tumors is shorter in length than cfDNA from healthy tissue. Finally, we characterized the genomic location of the detected CNAs to potentially enable the utility and interpretation of these results clinically. Collectively, the detected CNAs covered more than 95% of all autosomes, highlighting a benefit of using genome-wide sequencing. Taken together, these data demonstrate a proof-of-concept for using low-coverage, genome-wide sequencing of cfDNA in patients where tissue biopsy is not feasible or medically recommended. Citation Format: Taylor J. Jensen, Aaron M. Goodman, Shumei Kato, Christopher K. Ellison, Kimberly Kelly, Lisa Kim, Gregory A. Daniels, Kerry Fitzgerald, Erin McCarthy, Prachi Nakashe, Amin R. Mazloom, Graham McLennan, Eyad Almasri, Daniel S. Grosu, Marcia Eisenberg, Razelle Kurzrock. Genome-wide sequencing of cell-free DNA enables detection of copy number alterations in cancer patients where tissue biopsy is not feasible [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1362.

A critical stage of premalignant development in breast tumorigenesis is the emergence of preneoplastic cells that carry key oncogenic drivers yet remain quiescent. Preneoplastic cells maintain a state of quiescence, but may be triggered to proliferate through largely unknown factors. Expansion of these cells is key to driving clonal evolution, which is evident in early lesions and tissue fields surrounding tumors. Although preneoplastic development is determinant to successful evolution and formation of tumor growth, the cellular mechanisms controlling preneoplastic development is unclear. Our limited mechanistic understanding of quiescent, preneoplastic cell outgrowth highlights a major knowledge gap in the early control of tumor development. Advancements in detecting molecular changes in precancerous lesions have provided opportunities for devising early intervention strategies. Aberrant AKT activation has emerged as a founding oncogenic alteration in preneoplastic cells. While molecular changes predispose these cells to develop tumors and presumably favors their selection during early tumor development, how these alterations contribute to the cellular control of quiescent preneoplastic cells remains unknown. A major barrier to studying quiescent preneoplastic cells is the lack of physiologically relevant experimental models. Here, we utilized a novel quiescent, preneoplastic cell model that reconstructs the molecular and cellular contexts of preneoplastic breast tissue. We combined a three-dimensional (3D) organotypic cell culture on reconstituted basement membrane with cells that can be induced to activate defined oncogenic pathways. Using this model, we investigated the contribution of aberrant AKT activation in preneoplastic breast tumor development. We found that aberrant AKT signaling in quiescent, preneoplastic mammary cells does not stimulate cells to proliferate under normal conditions, but confers cell sensitivity to stroma-derived CXCL12 through upregulating the receptor CXCR4. Using heterotypic co-culture to recapitulate a tumor-promoting environment and quantitative proteomic analysis, we found that stromal-derived chemokine CXCL12 selectively promotes expansion of preneoplastic cells with hyperactivated AKT signaling. Mechanistically, we showed that AKT signaling induces the expression of the chemokine’s receptor, CXCR4, which confers the selective sensitivity to CXCL12. This CXCL12-CXCR4 axis serves as an alternate mitogenic signal through PKA and ERK to drive the downregulation of CDKN1C and promote cell proliferation in AKT-induced preneoplastic cells. Our studies uncovered a distinct role of AKT signaling in promoting preneoplastic development. Furthermore, our studies suggest a model of preneoplastic clonal expansion where oncogene signaling confers quiescent preneoplastic cell sensitivity to alternate mitogenic stimuli in a permissive tissue microenvironment. Citation Format: Zahra S. Masoud, Lisa Kim, Kelsey J. Weigel, Matthew M. Champion, Zachary T. Schafer, Cheuk Leung. CXCL12 drives selective expansion of AKT-induced quiescent, pre-neoplastic breast cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1996. doi:10.1158/1538-7445.AM2017-1996