Search

Inherited mutations in BRCA1, BRCA2, and PALB2 cause a high risk of breast cancer. Here, we conducted parallel conditional knockout (CKO) of Brca1, Palb2, and Brca2, individually and in combination, along with one copy of Trp53, in the mammary gland of nulliparous female mice. We observed a functional equivalence of the three genes in their basic tumor-suppressive activity, a linear epistasis of Palb2 and Brca2, but complementary roles of Brca1 and Palb2 in mammary tumor suppression, as combined ablation of either Palb2 or Brca2 with Brca1 led to delayed tumor formation. Whole-exome sequencing (WES) revealed both similarities and differences between Brca1 and Palb2 or Brca2 null tumors. Analyses of mouse mammary glands and cultured human cells showed that combined loss of BRCA1 and PALB2 led to high levels of reactive oxygen species (ROS) and increased apoptosis, implicating oxidative stress in the delayed tumor development in Brca1;Palb2 double CKO mice. The functional complementarity between BRCA1 and PALB2/BRCA2 and the role of ROS in tumorigenesis require further investigation.

Intratumor heterogeneity is an important mediator of poor outcomes in many cancers, including breast cancer. Genetic subclones frequently contribute to this heterogeneity; however, their growth dynamics and interactions remain poorly understood. PIK3CA and HER2 alterations are known to coexist in breast and other cancers. Herein, we present data that describe the ability of oncogenic PIK3CA mutant cells to induce the proliferation of quiescent HER2 mutant cells through a cell contact–mediated mechanism. Interestingly, the HER2 cells proliferated to become the major subclone over PIK3CA counterparts both in vitro and in vivo. Furthermore, this phenotype was observed in both hormone receptor–positive and –negative cell lines, and was dependent on the expression of fibronectin from mutant PIK3CA cells. Analysis of human tumors demonstrated similar HER2:PIK3CA clonal dynamics and fibronectin expression. Our study provides insight into nonrandom subclonal architecture of heterogenous tumors, which may aid the understanding of tumor evolution and inform future strategies for personalized medicine.

PURPOSE: Estrogen receptor-alpha (ER) is a therapeutic target of ER positive (ER+) breast cancers. Although ER signaling is complex, many mediators of this pathway have been identified. Specifically, phosphorylation of ER at serine 118 affects responses to estrogen and therapeutic ligands and has been correlated with clinical outcomes in ER+ breast cancer patients. We hypothesized that a newly described germline variant (S118P) at this residue would drive cellular changes consistent with breast cancer development and/or hormone resistance. METHODS: Isogenic human breast epithelial cell line models harboring ER S118P were developed via genome editing and characterized to determine the functional effects of this variant. We also examined the frequency of ER S118P in a case control study (N=536) of women with and without breast cancer with a familial risk. RESULTS: In heterozygous knock-in models, the S118P variant demonstrated no significant change in proliferation, migration, MAP Kinase pathway signaling, or response to the endocrine therapies tamoxifen and fulvestrant. Further, there was no difference in the prevalence of S118P between women with and without cancer relative to population registry databases. CONCLUSIONS: This study suggests that the ER S118P variant does not affect risk for breast cancer or hormone therapy resistance. Germline screening and modification of treatments for patients harboring this variant are likely not warranted.

Purpose: Although most human cancers display a single histology, there are unusual cases where two or more distinct tissue types present within a primary tumor. One such example is metaplastic breast carcinoma, a rare but aggressive cancer with a heterogeneous histology, including squamous, chondroid, and spindle cells. Metaplastic carcinomas often contain an admixed conventional ductal invasive or in situ mammary carcinoma component, and are typically triple-negative for estrogen receptor, progesterone receptor, and HER-2 amplification/overexpression. An unanswered question is the origin of metaplastic breast cancers. While they may arise independently from their ductal components, their close juxtaposition favors a model that postulates a shared origin, either as two derivatives from the same primary cancer or one histology as an outgrowth of the other. Understanding the mechanism of development of these tumors may inform clinical decisions. Experimental Design: We performed exome sequencing for paired metaplastic and adjacent conventional invasive ductal carcinomas in 8 patients and created a pipeline to identify somatic variants and predict their functional impact, without having normal tissue. We then determined the genetic relationships between the histologically distinct compartments. Results: In each case, the tumor components have nearly identical landscapes of somatic mutation, implying that the differing histologies do not derive from genetic clonal divergence. Conclusions: A shared origin for tumors with differing histologies suggests that epigenetic or noncoding changes may mediate the metaplastic phenotype and that alternative therapeutic approaches, including epigenetic therapies, may be required for metaplastic breast cancers. Clin Cancer Res; 23(16); 4875–84. ©2017 AACR.

The combined contributions of oncogenes and tumor suppressor genes toward carcinogenesis remain poorly understood. Elucidation of cancer gene cooperativity can provide new insights leading to more effective use of therapies. We used somatic cell genome editing to introduce singly and in combination PIK3CA mutations (E545K or H1047R) with TP53 alterations (R248W or knockout), to assess any enhanced cancerous phenotypes. The non-tumorigenic human breast epithelial cell line, MCF10A, was used as the parental cell line, and resultant cells were assessed via various in vitro assays, growth as xenografts, and drug sensitivity assays using targeted agents and chemotherapies. Compared to single-gene-targeted cells and parental controls, cells with both a PIK3CA mutation and TP53 alteration had increased cancerous phenotypes including cell proliferation, soft agar colony formation, aberrant morphology in acinar formation assays, and genomic heterogeneity. Cells also displayed varying sensitivities to anti-neoplastic drugs, although all cells with PIK3CA mutations showed a relative increased sensitivity to paclitaxel. All cell lines remained non-tumorigenic. This cell line panel provides a resource for further elucidating cooperative genetic mediators of carcinogenesis and response to therapies.

BACKGROUND/PURPOSE: TrkA overexpression occurs in over 20% of breast cancers, including triple-negative breast cancers (TNBC), and has recently been recognized as a potential driver of carcinogenesis. Recent clinical trials of pan-Trk inhibitors have demonstrated targeted activity against tumors harboring NTRK fusions, a relatively rare alteration across human cancers. Despite this success, current clinical trials have not investigated TrkA overexpression as an additional therapeutic target for pan-Trk inhibitors. Here, we evaluate the cancerous phenotypes of TrkA overexpression relative to NTRK1 fusions in human cells and assess response to pharmacologic Trk inhibition. EXPERIMENTAL DESIGN/METHODS: To evaluate the clinical utility of TrkA overexpression, a panel of TrkA overexpressing cells were developed via stable transfection of an NTRK1 vector into the non-tumorigenic breast cell lines, MCF10A and hTERT-IMEC. A panel of positive controls was generated via stable transfection with a CD74-NTRK1 fusion vector into MCF10A cells. Cells were assessed via various in vitro and in vivo analyses to determine the transformative potential and targetability of TrkA overexpression. RESULTS: TrkA overexpressing cells demonstrated transformative phenotypes similar to Trk fusions, indicating increased oncogenic potential. TrkA overexpressing cells demonstrated growth factor-independent proliferation, increased PI3Kinase and MAPKinase pathway activation, anchorage-independent growth, and increased migratory capacity. These phenotypes were abrogated by the addition of the pan-Trk inhibitor, larotrectinib. In vivo analysis demonstrated increased tumorgenicity and metastatic potential of TrkA overexpressing breast cancer cells. CONCLUSIONS: Herein, we demonstrate TrkA overexpressing cells show increased tumorgenicity and are sensitive to pan-Trk inhibitors. These data suggest that TrkA overexpression may be an additional target for pan-Trk inhibitors and provide a targeted therapy for breast cancer patients.

Purpose: Mutations in the estrogen receptor (ER)α gene, ESR1, have been identified in breast cancer metastases after progression on endocrine therapies. Because of limitations of metastatic biopsies, the reported frequency of ESR1 mutations may be underestimated. Here, we show a high frequency of ESR1 mutations using circulating plasma tumor DNA (ptDNA) from patients with metastatic breast cancer. Experimental Design: We retrospectively obtained plasma samples from eight patients with known ESR1 mutations and three patients with wild-type ESR1 identified by next-generation sequencing (NGS) of biopsied metastatic tissues. Three common ESR1 mutations were queried for using droplet digital PCR (ddPCR). In a prospective cohort, metastatic tissue and plasma were collected contemporaneously from eight ER-positive and four ER-negative patients. Tissue biopsies were sequenced by NGS, and ptDNA ESR1 mutations were analyzed by ddPCR. Results: In the retrospective cohort, all corresponding mutations were detected in ptDNA, with two patients harboring additional ESR1 mutations not present in their metastatic tissues. In the prospective cohort, three ER-positive patients did not have adequate tissue for NGS, and no ESR1 mutations were identified in tissue biopsies from the other nine patients. In contrast, ddPCR detected seven ptDNA ESR1 mutations in 6 of 12 patients (50%). Conclusions: We show that ESR1 mutations can occur at a high frequency and suggest that blood can be used to identify additional mutations not found by sequencing of a single metastatic lesion. Clin Cancer Res; 22(4); 993–9. ©2015 AACR.

BACKGROUND/PURPOSE: Tumor heterogeneity is a now well-recognized phenomenon that can affect the classification, prognosis and treatment of human cancers. Heterogeneity is often described in primary breast cancers based upon histologic subtypes, hormone- and HER2-receptor status, and immunolabeling for various markers, which can be seen within a single tumor as mixed cellular populations, or as separate discrete foci. EXPERIMENTAL DESIGN/METHODS: Here we present a case report of a patient’s primary breast cancer that had two separate but adjacent histologic components, one that was estrogen receptor (ER) positive, and the other ER negative. Each component was subjected to whole exome sequencing and compared for gene identity to determine clonal origin. RESULTS: Using prior bioinformatic tools, we demonstrated that both the ER positive and negative components shared many variants, including passenger and driver alterations. Copy number variations also supported the two components were derived from a single common clone. CONCLUSIONS: These analyses strongly suggest that the two ER components of this patient’s breast cancer were derived from the same clonal origin. Our results have implications for the evolution of breast cancers with mixed histologies, and how they might be best managed for optimal therapy.

It is now well established that DNA is shed or secreted into the circulation in both normal and disease states. However, the ability to exploit this knowledge for use in clinical medicine has only recently been made possible through the advent of new technologies. Currently, cell-free DNA is being developed as a “real-time liquid biopsy” biomarker to help guide clinical decisions in diverse fields such as prenatal screening, cancer, and solid organ transplantation. The ability of cell-free circulating tumor DNA to be measured quantitatively and qualitatively presents great opportunities in clinical oncology for using blood to monitor tumor burden and assess response to therapies. Here we provide a short review of historic and recent studies demonstrating the clinical potential of detecting and measuring cell-free DNA, with a specific focus on the use of circulating tumor DNA in oncology and cancer management.

Next-generation sequencing (NGS) is increasingly being used in cancer care to identify both somatic tumor driver mutations that can be targeted for therapy, and heritable mutations in the germline associated with increased cancer risk. This report presents a case of a JAK2 V617F mutation falsely identified as a duodenal cancer mutation via NGS. The patient was found to have a history of polycythemia vera, a disorder with a high incidence of JAK2 somatic mutations. Buccal cell DNA showed heterozygosity for the mutation, suggesting that it was potentially germline. However, subsequent resequencing of tumor, adjacent normal tissue, and fingernail DNA confirmed the mutation was somatic, and its presence in tumor and buccal cells resulted from contaminating blood cells. This report highlights important nuances of NGS that can lead to misinterpretation of results with potential clinical implications.