Mutation Profiling of Key Cancer Genes in Primary Breast Cancers and Their Distant Metastases.

Although the repertoire of somatic genetic alterations of primary breast cancers has been extensively catalogued, the genetic differences between primary and metastatic tumors have been less studied. In this study, we compared somatic mutations and gene copy number alterations of primary breast cancers and their matched metastases from patients with estrogen receptor (ER)-negative disease. DNA samples obtained from formalin-fixed paraffin-embedded ER-negative/HER2-positive (n = 9) and ER-, progesterone receptor (PR-), HER2-negative (n = 8) primary breast cancers and from paired brain or skin metastases and normal tissue were subjected to a hybridization capture-based massively parallel sequencing assay, targeting 341 key cancer genes. A large subset of nonsynonymous somatic mutations (45%) and gene copy number alterations (55%) was shared between the primary tumors and paired metastases. However, mutations restricted to either a given primary tumor or its metastasis, the acquisition of loss of heterozygosity of the wild-type allele, and clonal shifts of genes affected by somatic mutations, such as TP53 and RB1, were observed in the progression from primary tumors to metastases. No metastasis location-specific alterations were identified, but synchronous metastases showed higher concordance with the paired primary tumor than metachronous metastases. Novel potentially targetable alterations were found in the metastases relative to their matched primary tumors. These data indicate that repertoires of somatic genetic alterations in ER-negative metastatic breast cancers may differ from those of their primary tumors, even by the presence of driver and targetable somatic genetic alterations.Significance: Somatic genetic alterations in ER-negative breast cancer metastases may be distinct from those of their primary tumors, suggesting that for treatment-decision making, genetic analyses of DNA obtained from the metastatic lesion rather than from the primary tumor should be considered. Cancer Res; 78(12); 3112-21. ©2018 AACR.

Integrating Proteomics and Transcriptomics for Systematic Combinatorial Chimeric Antigen Receptor Therapy of AML.

Chimeric antigen receptor (CAR) therapy targeting CD19 has yielded remarkable outcomes in patients with acute lymphoblastic leukemia. To identify potential CAR targets in acute myeloid leukemia (AML), we probed the AML surfaceome for overexpressed molecules with tolerable systemic expression. We integrated large transcriptomics and proteomics datasets from malignant and normal tissues, and developed an algorithm to identify potential targets expressed in leukemia stem cells, but not in normal CD34+CD38- hematopoietic cells, T cells, or vital tissues. As these investigations did not uncover candidate targets with a profile as favorable as CD19, we developed a generalizable combinatorial targeting strategy fulfilling stringent efficacy and safety criteria. Our findings indicate that several target pairings hold great promise for CAR therapy of AML.

Genetic Heterogeneity in Therapy-Naïve Synchronous Primary Breast Cancers and Their Metastases.

Purpose: Paired primary breast cancers and metachronous metastases after adjuvant treatment are reported to differ in their clonal composition and genetic alterations, but it is unclear whether these differences stem from the selective pressures of the metastatic process, the systemic therapies, or both. We sought to define the repertoire of genetic alterations in breast cancer patients with de novo metastatic disease who had not received local or systemic therapy.Experimental Design: Up to two anatomically distinct core biopsies of primary breast cancers and synchronous distant metastases from nine patients who presented with metastatic disease were subjected to high-depth whole-exome sequencing. Mutations, copy number alterations and their cancer cell fractions, and mutation signatures were defined using state-of-the-art bioinformatics methods. All mutations identified were validated with orthogonal methods.Results: Genomic differences were observed between primary and metastatic deposits, with a median of 60% (range 6%-95%) of shared somatic mutations. Although mutations in known driver genes including TP53, PIK3CA, and GATA3 were preferentially clonal in both sites, primary breast cancers and their synchronous metastases displayed spatial intratumor heterogeneity. Likely pathogenic mutations affecting epithelial-to-mesenchymal transition-related genes, including SMAD4, TCF7L2, and TCF4 (ITF2), were found to be restricted to or enriched in the metastatic lesions. Mutational signatures of trunk mutations differed from those of mutations enriched in the primary tumor or the metastasis in six cases.Conclusions: Synchronous primary breast cancers and metastases differ in their repertoire of somatic genetic alterations even in the absence of systemic therapy. Mutational signature shifts might contribute to spatial intratumor genetic heterogeneity. Clin Cancer Res; 23(15); 4402-15. ©2017 AACR.

Genetic analysis of microglandular adenosis and acinic cell carcinomas of the breast provides evidence for the existence of a low-grade triple-negative breast neoplasia family.

Acinic cell carcinoma is an indolent form of invasive breast cancer, whereas microglandular adenosis has been shown to be a neoplastic proliferation. Both entities display a triple-negative phenotype, and may give rise to and display somatic genomic alterations typical of high-grade triple-negative breast cancers. Here we report on a comparison of previously published data on eight carcinoma-associated microglandular adenosis and eight acinic cell carcinomas subjected to targeted massively parallel sequencing targeting all exons of 236 genes recurrently mutated in breast cancer and/or DNA repair-related. Somatic mutations, insertions/ deletions, and copy number alterations were detected using state-of-the-art bioinformatic algorithms. All cases were of triple-negative phenotype. A median of 4.5 (1-13) and 4.0 (1-7) non-synonymous somatic mutations per carcinoma-associated microglandular adenosis and acinic cell carcinoma were identified, respectively. TP53 was the sole highly recurrently mutated gene (75% in microglandular adenosis versus 88% in acinic cell carcinomas), and TP53 mutations were consistently coupled with loss of heterozygosity of the wild-type allele. Additional somatic mutations shared by both groups included those in BRCA1, PIK3CA, and INPP4B. Recurrent (n=2) somatic mutations restricted to microglandular adenosis or acinic cell carcinomas included those affecting PTEN and MED12 or ERBB4, respectively. No significant differences in the repertoire of somatic mutations were detected between microglandular adenosis and acinic cell carcinomas, and between this group of lesions and 77 triple-negative carcinomas from The Cancer Genome Atlas. Microglandular adenosis and acinic cell carcinomas, however, were genetically distinct from estrogen receptor-positive and/or HER2-positive breast cancers from The Cancer Genome Atlas. Our findings support the contention that microglandular adenosis and acinic cell carcinoma are part of the same spectrum of lesions harboring frequent TP53 somatic mutations, and likely represent low-grade forms of triple-negative disease with no/minimal metastatic potential, of which a subset has the potential to progress to high-grade triple-negative breast cancer.

Massively parallel sequencing analysis of synchronous fibroepithelial lesions supports the concept of progression from fibroadenoma to phyllodes tumor.

Phyllodes tumors (PTs) and fibroadenomas (FAs) are fibroepithelial lesions (FELs) of the breast. Although mutations affecting exon 2 of MED12 are highly recurrent in FAs and PTs, TERT promoter hotspot mutations are frequently found in PTs but are vanishingly rare in FAs. Malignant transformation of benign PTs is well-documented, but the progression from FA to PT remains a matter of contention. Here we report on the somatic genetic alterations in multiple ipsilateral synchronous FELs (three FAs, one benign PT, and one malignant PT) occurring in the same patient. DNA samples extracted from each tumor and matched normal tissue were subjected to targeted massively parallel sequencing using the Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT) assay. This analysis revealed MED12 mutations in all lesions. One FA and the benign PT harbored a MED12Gly44Val mutation, whereas another FA and the malignant PT displayed a MED12Gly44Asp mutation. The remaining FA had an independent distinct MED12Gly44Cys mutation. A formal clonality analysis suggested a clonal relationship between the FELs with identical MED12 mutations (P<0.05). A clonal TERT promoter hotspot mutation was identified exclusively in the malignant PT. The identification of distinct MED12 mutations in multifocal ipsilateral and synchronous FELs supports the notion that co-existing mammary fibroepithelial tumors can arise independently. Conversely, the co-existence of identical MED12 mutations indicates clonal relatedness among FAs and PTs, corroborating the hypothesis that FAs may constitute the substrate from which PTs develop. Our findings also support the notion that acquisition of TERT promoter mutations may drive the progression of FELs.

Quantitative assessment of intragenic receptor tyrosine kinase deletions in primary glioblastomas: their prevalence and molecular correlates.

Intragenic deletion is the most common form of activating mutation among receptor tyrosine kinases (RTK) in glioblastoma. However, these events are not detected by conventional DNA sequencing methods commonly utilized for tumor genotyping. To comprehensively assess the frequency, distribution, and expression levels of common RTK deletion mutants in glioblastoma, we analyzed RNA from a set of 192 glioblastoma samples from The Cancer Genome Atlas for the expression of EGFRvIII, EGFRvII, EGFRvV (carboxyl-terminal deletion), and PDGFRAΔ8,9. These mutations were detected in 24, 1.6, 4.7, and 1.6 % of cases, respectively. Overall, 29 % (55/189) of glioblastomas expressed at least one RTK intragenic deletion transcript in this panel. For EGFRvIII, samples were analyzed by both quantitative real-time PCR (QRT-PCR) and single mRNA molecule counting on the Nanostring nCounter platform. Nanostring proved to be highly sensitive, specific, and linear, with sensitivity comparable or exceeding that of RNA seq. We evaluated the prognostic significance and molecular correlates of RTK rearrangements. EGFRvIII was only detectable in tumors with focal amplification of the gene. Moreover, we found that EGFRvIII expression was not prognostic of poor outcome and that neither recurrent copy number alterations nor global changes in gene expression differentiate EGFRvIII-positive tumors from tumors with amplification of wild-type EGFR. The wide range of expression of mutant alleles and co-expression of multiple EGFR variants suggests that quantitative RNA-based clinical assays will be important for assessing the relative expression of intragenic deletions as therapeutic targets and/or candidate biomarkers. To this end, we demonstrate the performance of the Nanostring assay in RNA derived from routinely collected formalin-fixed paraffin-embedded tissue.

The somatic genomic landscape of glioblastoma.

We describe the landscape of somatic genomic alterations based on multidimensional and comprehensive characterization of more than 500 glioblastoma tumors (GBMs). We identify several novel mutated genes as well as complex rearrangements of signature receptors, including EGFR and PDGFRA. TERT promoter mutations are shown to correlate with elevated mRNA expression, supporting a role in telomerase reactivation. Correlative analyses confirm that the survival advantage of the proneural subtype is conferred by the G-CIMP phenotype, and MGMT DNA methylation may be a predictive biomarker for treatment response only in classical subtype GBM. Integrative analysis of genomic and proteomic profiles challenges the notion of therapeutic inhibition of a pathway as an alternative to inhibition of the target itself. These data will facilitate the discovery of therapeutic and diagnostic target candidates, the validation of research and clinical observations and the generation of unanticipated hypotheses that can advance our molecular understanding of this lethal cancer.