OP-JNCI190159 107..110

Chemotherapy and radiation therapy are the foundations of adjuvant therapy for early-stage breast cancer. As a complication of cytotoxic regimens, breast cancer patients are at risk for therapy-related myeloid neoplasms (t-MNs). These t-MNs are commonly refractory to antileukemic therapies and result in poor patient outcomes. We previously demonstrated that somatic mutations in leukemia-related genes are present in the tumor-infiltrating leukocytes (TILeuks) of a subset of early breast cancers. Here, we performed genomic analysis of microdissected breast cancer tumor cells and TILeuks from seven breast cancer patients who subsequently developed leukemia. In four patients, mutations present in the leukemia were detected in breast cancer TILeuks. This finding suggests that TILeuks in the primary breast cancer may harbor the ancestor of the future leukemogenic clone. Additional research is warranted to ascertain whether infiltrating mutant TILeuks could constitute a biomarker for the development of t-MN and to determine the functional consequences of mutant TILeuks. Therapy-related myeloid neoplasms (t-MNs), including acute myeloid leukemia (AML), develop in a subset of early-stage breast cancer patients treated with radiation therapy and chemotherapy. These t-MNs are commonly refractory to antileukemic therapies, resulting in poor patient outcomes (1). Breast cancer is the most frequent solid tumor for which t-MN is observed, accounting for 70% of t-MN cases among women (2–4). Clonal hematopoiesis is the clonal expansion of hematopoietic stem and progenitor cells characterized by common somatic mutations, most often in genes with a known role in myeloid malignancies. It is associated with an increased risk for hematologic malignancies and more common in patients who have a history of a solid tumor (5,6). We previously demonstrated that leukocytes harboring somatic oncogenic mutations affecting leukemia-related genes, including TET2, BCOR, and EZH2, infiltrate nearly 50% of primary breast cancers (7) and that tumor-infiltrating leukocytes (TILeuks) with mutations in clonal hematopoiesis genes can be enriched in breast cancers before the detection of clonal hematopoiesis in the blood (7). Here, we sought to define whether t-MNs following breast cancer treatment were clonally related to TILeuks present in the antecedent primary breast cancer (Supplementary Methods, available online) by examining tumor samples from individuals with a history of breast cancer who developed acute leukemia. We identified seven patients who developed t-MN after breast cancer treatment. All patients provided written consent as part of an approved protocol by the institutional review board of Memorial Sloan Kettering Cancer Center. Patients underwent surgery for their breast cancer and received adjuvant or B R IE F C O M M U N IC A T IO N Received: March 25, 2019; Revised: June 10, 2019; Accepted: August 6, 2019 © The Author(s) 2019. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com 107 JNCI J Natl Cancer Inst (2020) 112(1): djz157 doi: 10.1093/jnci/djz157 First published online August 27, 2019 Brief Communication neoadjuvant chemotherapy and radiation before their leukemia diagnosis. All patients but one received adjuvant breast chemotherapy for their breast cancer (patient 2 received neoadjuvant therapy; patient 6 received radioactive iodine 2 years before breast cancer diagnosis for thyroid cancer; Supplementary Methods, available online). We screened for evidence of t-MN following breast cancer using these criteria: less than 20% blast count (bone marrow or peripheral blood leukocytes), access to frozen mononuclear cells or bone marrow aspirate slides prepared at the time of leukemia diagnosis, and histologic blocks of the primary breast cancer suitable for laser capture microdissection (LCM). Breast cancer samples for all the patients were obtained from primary breast cancer surgical specimens. For each breast cancer patient, we sequenced the secondary leukemia samples using a targeted sequencing panel of 585 genes commonly mutated in leukemia, lymphoma, and solid tumors (HemePACT, Supplementary Table 1, available online) (8), and then used LCM to isolate TILeuks and breast tumor cells for sequencing. For each oncogenic mutation and structural variant identified in the leukemia sample, we performed targeted amplicon sequencing (Supplementary Methods, available online) to confirm the absence or presence of potentially oncogenic mutations and structural variants in the two compartments of the matching breast cancer samples (Figure 1; Supplementary Table 2, available online). Four of the seven patients (patients 2, 4, 6, and 7) had mutations present both in the t-MN and in the preleukemia breast cancer TILeuks collected before the diagnoses of leukemia (respectively, 6, 6, 5, and 2 years earlier; Figure 2). We evaluated the statistical significance of enrichment of mutations in TILeuk vs breast tumor cells using a two-tailed v test with a P value cutoff of . 05 (Supplementary Table 2, available online). KRAS GRIN2A GATA2 GNAQ TP53 ASXL1 NOTCH1 JAK3 BRD4 FLT3 WT1 STAG2 KMT2D CEBPA SDHA TET2 IDH2 TGFBR2 DNMT3A M S K 1