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Background. The female mammary gland can develop a myriad of epithelial proliferative lesions including, high risk lesions, in-situ and invasive carcinomas. Identification of these pre-neoplastic and neoplastic conditions in biopsy specimens is crucial for proper patient management and may sometimes pose diagnostic challenges for pathologists. Recent research has shown that machine learning algorithms applied to whole slide images (WSI) can accurately detect and grade various cancers; herein, we devise and test a system that classifies the most common preneoplastic and neoplastic conditions of the female breast from WSIs. Design. De-identified slides were scanned on Leica AT2 whole slide scanners (20x; 0.5 µm/pixel) from MSK database were retrieved. Clinical diagnostic metadata were extracted from the pathology reports. Using a multi-label multiple-instance learning (ML-MIL) approach, a SE-ResNet50 Convolutional Neural Network (CNN) was trained to classify atypical lobular hyperplasia (ALH), atypical ductal hyperplasia (ADH), lobular carcinoma in situ (LCIS), ductal carcinoma in situ (DCIS), invasive lobular carcinoma (ILC), invasive ductal carcinoma (IDC). In additional morphological subtypes including apocrine, mucinous, solid papillary, micropapillary, and tubular carcinoma were trained. The system uses the WSI as an input and outputs a slide level class and heatmap for the presence of the trained classes. A validation dataset separate from the training set was used to assess performance of the trained model. Results. The CNN was trained on 9,751 surgical specimens (biopsy, 6,289; excision, 3,462) comprising 40,637 slides. The system was validated on 3,183 breast specimens (biopsy, 1,934; excision, 1,249) comprising 11,447 digital slides that were not included in the training of the CNN model. Validation performance in terms of Area Under Receiver Operating Characteristic Curve (AUROC) for each class is shown in Table 1. Conclusion. The trained CNN had a high performance in identifying the presence of ADH, ALH, DCIS, IDC, ILC, LCIS, and, apocrine, micropapillary, mucinous, solid papillary, and tubular carcinomas. Further studies expanding classes to include all clinically relevant lesions and morphologies are underway. In addition, the same approach can be used to detect microinvasions and calcifications in breast tissue. Table 1.Area Under Receiver Operating Characteristic Curve for Breast Lesion ClassesClassNum. Positive (specimens)Num. Negative (specimens)AUROCADH24718640.903ALH20918860.950LCIS17520080.958DCIS81920920.956IDC52126620.956ILC7131120.934Apocrine2431590.931Micropapillary17230110.927Mucinous1231710.994Solid Papillary1531680.908Tubular carcinoma831750.990 Citation Format: Matthew G Hanna, Patricia Raciti, Alican Bozkurt, Ran Godrich, Julian Viret, Donghun Lee, Philippe Mathieu, Matthew Lee, Eugene Vorontsov, Tomer Sabo, Felipe C Geyer, Jorge S Reis-Filho, Leo Grady, Thomas Fuchs, Christopher Kanan. Subtyping invasive carcinomas and high-risk lesions for machine learning based breast pathology [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD11-02.

Introduction: Metaplastic breast carcinomas (MBCs) and uterine carcinosarcomas (UCSs) are histologically similar, being often characterized by an admixture of adenocarcinoma areas with areas displaying sarcomatoid differentiation. We sought to investigate whether their histologic similarities would be paralleled by similar patterns of genetic alterations, and to determine whether the different histologic components of MBCs and UCSs would be clonally related. Methods: Whole exome sequencing (WES) data from 35 MBCs previously analyzed by our group and 57 UCSs from The Cancer Genome Atlas (TCGA) study were reanalyzed. Somatic single nucleotide variants were detected with MuTect and indels with Strelka, Varscan2, Scalpel and Lancet. Copy number alterations were inferred using FACETS and functional annotation of the non-synonymous somatic mutations, amplifications or homozygous deletions was performed. We further microdissected the histologically distinct components of 11 MBCs and six UCSs and subjected each component to WES. Clonal decomposition was performed using PyClone. Results: The most frequent somatic mutations identified in MBCs were TP53 (69%), PIK3CA (29%), FAT3 (26%) and PTEN (14%), whereas the most frequently mutated genes in UCSs were TP53 (84%), FBXW7 (35%), PIK3CA (29%), PTEN (15%) and PPP2R1A (15%). MBCs displayed a significantly higher frequency of mutations targeting FAT3 (26% vs 4%, P Conclusions: Our findings support the contention that UCSs constitute the uterine counterpart of MBCs due to their similar histology and patterns of genetic alterations affecting the same signaling pathways (i.e. TP53, PI3K, Wnt and Notch). In each MBC and UCS analyzed here, the histologically distinct components were found to be clonally related. Citation Format: Da Cruz Paula A, Brown D, Geyer FC, Smith E, Pareja F, Papanastasiou AD, Fusco N, Marchio C, Brogi E, Wen HY, Vincent-Salomon A, Norton L, Weigelt B, Reis-Filho JS. Metaplastic breast carcinomas and uterine carcinosarcomas are histologically and genetically related [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-07-02.

Introduction: Mucinous carcinoma of the breast (MCB) is a rare histologic subtype of estrogen receptor (ER)-positive invasive carcinoma, and is characterized by tumor cells floating in pools of mucin. Despite their characteristic histology, MCBs are heterogeneous at the genetic level and no driver genetic alterations have been identified. Notably, MCBs lack the common genetic alterations found in ER-positive invasive ductal carcinomas (i.e. 16q losses, 1q gains and PIK3CA mutations). Fusion genes have been reported in breast cancer, including highly recurrent or pathognomonic fusions genes, such as the ETV6-NTRK3 and MYB/MYBL1 rearrangements in secretory carcinoma and adenoid cystic carcinoma, respectively. In this study we sought to define whether MCBs would be underpinned by a pathognomonic fusion gene. Materials and methods: Seven pure mucinous A (hypocellular), seven pure mucinous B (hypercellular), and the mucinous component of a mixed MCB were microdissected, and subjected to RNA extraction followed by RNA-sequencing for fusion gene discovery. Read pairs supporting chimeric transcripts were identified using INTEGRATE, FusionCatcher and STARfusion. The Bayesian driver probability of the candidate fusion genes was annotated using OncoFuse. In-frame fusion gene candidates with a high driver probability were validated using orthogonal methods (RT-PCR and Sanger sequencing). Results: Our analysis identified fusion genes in 47% (7/15) of the MCBs analyzed (29% (2/7) of type A MCBs, 57% (4/7) type B MCBs and in the mucinous component of one mixed MCB). The OAZ1-CSNK1G2 and RFC4-LPP fusion genes were identified in 20% (3/15) and 13% (2/15) of the cases, respectively. The OAZ1-CSNK1G2 chimeric transcript results in the truncation of the kinase domain of CSNK1G2, which represses ER transactivation. The RFC4-LPP chimeric transcript leads to the fusion of exons 1-3 of RFC4 and exons 5-11 of LPP, where the LIM domains of LPP are conserved. LPP is a known partner of fusion genes identified in mesenchymal tumors and reported to mediate TGF-β induced breast oncogenesis via its LIM domain. Additional validated, potentially pathogenic fusion genes identified in MCBs involved kinases, phosphatases or regulators of tyrosine kinase receptor signaling, such as IRAK3-PPM1H (n=1), GIGYF2–GFRA3 (n=1) and PHF20-FAM217B (n=1). Conclusions: MCBs harbor fusion genes in almost half of the cases with two fusions being recurrent, involving primarily genes encoding kinases, phosphatases or receptor tyrosine kinase signaling regulators. Nevertheless, due to the lack of recurrence of a specific fusion gene, this special histologic type of breast cancer is unlikely to be underpinned by a highly recurrent/ pathognomonic pathogenic fusion gene. Citation Format: Pareja F, Gularte-Merida R, Da Cruz Paula A, Brown D, Geyer FC, Piscuoglio S, Marchio C, Vincent-Salomon A, Brogi E, Weigelt B, Reis-Filho JS. Recurrent but not pathognomonic fusion genes in mucinous carcinomas of the breast [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-06-07.

Introduction: Mucinous (MBCs) and neuroendocrine breast carcinomas (NBCs) are special histologic types of estrogen receptor (ER)-positive breast cancers. Together, MBCs and NBCs account for approximately 5% of invasive breast cancers. NBCs are defined by the expression of neuroendocrine markers (i. e., chromogranin and/ or synaptophysin), whereas MBCs consist of nests of cancer cells floating in pools of extracellular mucin and can be either hypocellular/type A (MBC-A) or hypercellular/type B (MBC-B). MBC-B may display neuroendocrine differentiation. We compared the repertoire of somatic genetic alterations in MBCs-A and MBCs-B, and in NBCs, and evaluated whether they differ from ER-positive/ HER2-negative invasive ductal carcinomas of no special type (IDC-NSTs). Materials and methods: Reanalysis of the targeted capture or whole exome sequencing data of 22 MBCs (12 MBCs-A and 10 MBCs-B), and of 15 NBCs was performed. The BAM files were retrieved and somatic mutations were detected with MuTect, indels with Strelka, Varscan2, Scalpel and Lancet, and copy number alterations using FACETS. The mutational repertoire of MBCs-A and MCBs-B was compared to that of NBCs, and of ER-positive/ HER2-negative IDC-NSTs from The Cancer Genome Atlas (TCGA) breast cancer study (n=310). Results: The most frequently mutated genes in MBCs-A were SF3B1 and FRG1B (17%, each); in MBCs-B were GATA3 and KMT2C (30%, each), and in NBCs were FOXA1 and TBX3 (20%, each) and KMT2C (20%). No significant differences were observed in single gene comparisons between MBCs-A, MBCs-B and NBCs (Fisher's exact tests, p>0.05). When compared with ER-positive/HER2-negative IDC-NSTs from TCGA, NBCs harbored a higher frequency of mutations targeting FOXA1 (20% vs 2.9%, Fisher's exact test, p0.05). Concurrent 1q gains and 16q losses, the hallmark copy number alterations of ER-positive/HER2-negative breast cancer were found in 47% of the ER-positive/HER2-negative IDC-NSTs from TCGA, but only present in 20% of NBCs and in none of the MBCs-A or MBCs-B. Conclusion: Type A MBCs, type B MBCs and NBCs harbor similar patterns of genetic alterations, including a low frequency of PIK3CA mutations and of concurrent 1q gains/ 16q deletions. Taken together, our data suggest that both MBCs and the vast majority of NBCs constitute a spectrum of histologically and genomically related subtypes, distinct from ER-positive/HER2-negative IDC-NSTs. Citation Format: Pareja F, Da Cruz Paula A, Selenica P, Lee JY, Marchio C, Brown D, Geyer FC, Brogi E, Vincent-Salomon A, Weigelt B, Reis-Filho JS. Mucinous and neuroendocrine breast cancers: A spectrum of genetically-related lesions distinct from common forms of estrogen receptor-positive breast cancers [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-06-08.

Introduction:Pathogenic and/or founder germline variants in the ataxia-telangiectasia mutated (ATM) gene confer an increased breast cancer (BC) risk. The protein kinase ATM plays a central role inDNA double-strand break-repair and in the activation of downstream targets such as p53 and BRCA1. We sought to define the repertoire of somatic genetic alterations of BCs from patients with pathogenic germline ATM mutations and whether somatic loss of heterozygosity (LOH) of ATM would be present in these cancers. Methods: 21 BCs from ATM germline mutation carriers were microdissected. Tumor and normal DNA samples were subjected to whole-exome sequencing (WES, n=12) or massively parallel sequencing targeting all coding regions and selected intronic and regulatory regions of 410 key cancer genes (n=9). Somatic mutations, copy number alterations, cancer cell fractions, large-scale state transitions (LSTs) and mutational signatures were defined using state-of-the-art bioinformatics algorithms. ABSOLUTE and FACETS were employed to assess LOH of the wild-type allele of ATM. Results: Of the patients included in this study, 71%, 24% and 5% of cases harbored ATM missense (all but one p.V2424G), frame-shift and nonsense germline mutations, respectively. All tumors were ER-positive and four (19%) were HER2-positive. The median age of the patients was 46 years (32–79 years). Our analyses revealed biallelic inactivation of ATM through LOH of the wild-type allele in 16 of 21 cases (76%), and second somatic ATM mutations were not found. The median number of non-synonymous somatic mutations was 38 (range 15-113) and 2 (range 0-8)in tumors subjected to WES and targeted sequencing, respectively. The repertoire of somatic genetic alterations of ATM-associated BCs was found to be heterogeneous, including clonal PIK3CA mutations (24%), GATA3 mutations (19%), FANCI amplifications (19%) and CCND1 amplifications (14%). Importantly, however, no somatic mutations affecting TP53 were found. Analysis of the WES data revealed that 5 (42%) ATM-associated BCs displayed high LST scores, all of which harbored bi-allelic ATM inactivation. In contrast to BRCA1- and BRCA2-associated BCs, which frequently display the mutational signature 3 associated with defective homologous recombination DNA repair, the ATM-associated BCs studied displayed the ageing mutational signature (i.e. signature 1). Comparison of the mutational profiles of the ATM--associated BCs subjected to WES (n=12) with those of BRCA1- (n=11) and BRCA2-associated (n=10) BCs from The Cancer Genome Atlas revealed that TP53 was more frequently mutated in BCs from BRCA1 germline mutation carriers (0% vs 72%, P Conclusion: ATM-associated BCs frequently display bi-allelic ATM inactivation through LOH of the wild-type allele and a subset of these cases displayed high levels of LSTs. These findings suggest that at least in a subset of ATM-associated BCs, biallelic inactivation of ATM rather than a dominant negative effect of the germline mutation may be the mechanism of inactivation of this tumor suppressor gene. The repertoire of somatic genetic alterations of ATM-associated BCs is heterogeneous, with a noticeable lack of TP53 somatic mutations. Citation Format: Weigelt B, Bi R, Kumar R, James PA, Thorne H, Couch FJ, Eccles DM, Blows F, Geyer FC, Li A, Selenica P, Lim RS, Blecua P, Shen R, Wen H, Robson ME, Reis-Filho JS, Chenevix-Trench G. The landscape of somatic genetic alterations in breast cancers from ATM germline mutation carriers [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD1-15.

Introduction:Adenomyoepithelioma (AME) of the breast is a rare biphasic tumor, characterized by epithelial and myoepithelial differentiation. Although AMEs have an indolent clinical course, a subset may progress to carcinoma and metastasize. We sought to define the mutational landscape of AMEs and investigate the functional impact of recurrent pathogenic mutations identified in these tumors. Methods: Thirty-one AMEs were subjected to whole-exome sequencing (WES, n=8) or massively parallel sequencing targeting all coding regions of 410 key cancer genes and intronic and regulatory regions of selected genes (n=23). Somatic genetic alterations were defined using state-of-the-art bioinformatics algorithms. In an additional set of 12 AMEs, Sanger sequencing analysis of HRAS, PIK3CA and AKT1 was performed. Non-tumorigenic estrogen receptor (ER)-negative mammary epithelial cells (i.e. MCF10A, MCF10A with a PIK3CA H1047R mutation knock-in and MCF12A) were utilized for functional studies using both conventional monolayer and three-dimensional (3D) culture assays. Results: 27 (63%) and 16 (37%) AMEs were ER-positive and ER-negative, respectively. ER-negativity was significantly associated with histologic features predictive of a more aggressive behavior, with a higher number of mutations and copy number alterations, and with a distinct mutational profile as compared to ER-positive AMEs. Of the 27 ER-positive AMEs, 12 cases (44%) harbored PIK3CA hotspot mutations, and 5 PIK3CA wild-type cases displayed E17K AKT1 hotspot mutations. By contrast, of the 16 ER-negative AMEs, 9 (56%), 9 (56%) and 3 (19%) harbored HRAS, PIK3CA (mostly E545K and H1047R hotspots) and PIK3R1 mutations, respectively. Strikingly, all HRAS mutations were restricted to ER-negative AMEs, affected the hotspot codon Q61 (Q61R/K), and all but one co-occurred with PIK3CA or PIK3R1 mutations. In addition, HRAS Q61 hotspot mutations were significantly associated with necrosis (p=0.01) and high mitotic rates (p=0.03). CDKN2A homozygous deletions were also detected only in ER-negative AMEs (19%) and found to be significantly associated with progression to carcinoma (p=0.001). Forced expression of HRAS Q61R in MCF10A and MCF12A cells resulted in i) increased proliferation and transformation, ii) an irregular growth pattern in 3D organotypic cell cultures, iii) partial loss of the epithelial phenotype, and iv) acquisition of myoepithelial differentiation, which was more overt in PIK3CA-mutant MCF10A cells. HRAS Q61Rinduced hyperactivation of the PI3K pathway, but both PI3K and MAPK pathways likely contributed to the RAS-mediated proliferation, which was completely arrested by combined AKT and MEK inhibition. Conclusion: AMEs are phenotypically and genetically heterogeneous. Whilst pathogenic mutations in PI3K pathway-related genes occur across the spectrum of lesions, HRAS Q61 hotspot mutations are restricted to ER-negative AMEs. Our genomic and functional analyses indicate that HRAS Q61 mutations are driver events in the pathogenesis of ER-negative AMEs and, in conjunction with mutant PIK3CA, may lead to the acquisition of myoepithelial differentiation in breast epithelial cells. Citation Format: Geyer FC, Li A, Papanastasiou AD, Smith A, Selenica P, Burke KA, Edelweiss M, Wen H-C, Piscuoglio S, Schultheis AM, Martelotto LG, Pareja F, Kumar R, Brandes A, Lozada J, Macedo GS, Muenst S, Terracciano LM, Jungbluth A, Foschini MP, Wen HY, Brogi E, Palazzo J, Rubin BP, Ng CKY, Norton L, Varga Z, Ellis IO, Rakha E, Chandarlapatty S, Weigelt B, Reis-Filho JS. Estrogen receptor-negative breast adenomyoepitheliomas are driven by co-occurring HRAS hotspot and PI3K pathway gene mutations: A genetic and functional analysis [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD4-13.

Introduction: Mucinous carcinoma of the breast (MCB) is a rare histologic form of estrogen receptor (ER)-positive invasive carcinoma, accounting for up to 2% of breast cancers. MCBs are characterized by clusters of tumor cells floating in lakes of extracellular mucin, and are classified into mucinous A (paucicellular) and mucinous B (hypercellular) subtypes. Some MCBs are found admixed with invasive ductal carcinoma components, and then classified as mixed MCBs. The aims of this study were to determine the repertoire of somatic mutations of MCBs and to ascertain whether these genetic alterations are distinct from those identified in common forms of ER+/HER2- invasive breast cancers (IBCs). We also sought to determine whether the mucinous and ductal components of mixed MCBs would be clonally related. Materials and methods: Thirty MCBs including 25 pure MCBs (n=13 mucinous A, n=12 mucinous B) and five mixed MCBs were microdissected and subjected to whole exome sequencing. Each tumor component of mixed cases was microdissected and analyzed separately. Somatic mutations, copy number alterations and mutational signatures were defined using state-of-the-art bioinformatics methods. The mutational repertoire of MCBs was compared with that of ER+/HER2- IBCs (n = 240) from The Cancer Genome Atlas (TCGA) breast cancer study. Results: The genes most frequently mutated in MCBs were GATA3 (27%, 8/30, all frameshift mutations), KMT2C (13%, 4/30) and MAP3K1 (10%, 3/30). No significant differences were identified in single gene comparisons between mucinous A and mucinous B MCBs or between pure MCBs and the mucinous component of mixed MCBs (Fisher's exact tests, p>0.05). As compared to common forms of ER+/HER2- IBC, MCBs had a lower frequency of PIK3CA mutations (7% vs 42%, p Conclusions: The repertoire of somatic mutations in MCBs is distinct from that of common forms of ER+/HER2- IBCs. These differences include the lack of concurrent 1q gains/16q losses, a lower frequency of PIK3CA mutations and a higher frequency of GATA3 mutations in pure MCBs. Citation Format: Pareja F, Geyer FC, Piscuoglio S, Selenica P, Kumar R, Lim RS, Guerini-Rocco E, Marchio C, Mariani O, Ng CKY, Brogi E, Norton L, Vincent-Salomon A, Weigelt B, Reis-Filho JS. Mucinous breast carcinomas: A genomically distinct subtype of estrogen receptor-positive invasive breast cancers [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-05-08.

Introduction: Breast adenoid cystic carcinoma (AdCC) is a rare type of triple-negative breast cancer associated with an indolent clinical behavior. AdCCs provide a clear example of genotypic-phenotypic correlation with the majority harboring the MYB-NFIB fusion gene. In this study, we sought to identify alternative driver genetic alterations in breast AdCCs lacking the MYB-NFIB fusion gene. Methods: Nucleic acids obtained from four breast AdCCs lacking the MYB-NFIB fusion gene as defined by reverse transcription (RT)-PCR and/or fluorescence in situ hybridization (FISH) were subjected to RNA-sequencing (n=3), whole-genome (n=2) and/or targeted (n=1) massively parallel sequencing. Sequencing data were analyzed using state-of-the-art bioinformatics algorithms, and potential alternative driver genetic alterations were validated using orthogonal sequencing and molecular pathology methods. Results: RNA-sequencing revealed the presence of MYBL1-ACTN1 or MYBL1-NFIB fusion genes in two breast AdCCs, which were validated by whole-genome sequencing and/or MYBL1 FISH analysis. Both MYBL1 fusion gene-positive cases were found to overexpress MYBL1 as defined by quantitative RT-PCR analysis. In the third MYB-NFIB-negative breast AdCC studied, a high-level MYB gene amplification coupled with overexpression of MYB at the mRNA and protein levels was identified. In the fourth breast AdCC, which expressed high levels of MYB, whole-genome and RNA-sequencing revealed no definite alternative driver alteration, however, a MYBL2 intronic mutation was found in this case, which was associated with high levels of MYBL2 mRNA expression. In this case, single sample gene set enrichment analysis revealed activation of pathways similar to those activated in AdCCs harboring the MYB-NFIB or MYBL1 fusions genes. Conclusion: We demonstrate that in breast AdCCs lacking the MYB-NFIB fusion gene MYBL1 rearrangements and MYB amplification are likely alternative driver genetic events. Given that activation of MYB/MYBL1 and their downstream targets can be driven by the MYB-NFIB fusion gene, MYBL1 rearrangements, MYB amplification or other yet to be validated mechanisms (e.g. MYBL2 non-coding mutations), our findings further suggest that breast AdCCs constitute a convergent phenotype. Citation Format: Kim J, Geyer FC, Martelotto LG, Ng CKY, Lim RS, Selenica P, Li A, Pareja F, Fusco N, Edelweiss M, Mariani O, Badve S, Vincent-Salomon A, Norton L, Reis-Filho JS, Weigelt B. Novel driver genetic alterations in MYB-NFIB-negative breast adenoid cystic carcinomas [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-05-03.

Introduction: Metaplastic breast carcinoma (MBC) is a rare histologic type of triple-negative breast cancer (TNBC), characterized by the presence of cells displaying squamous and/or mesenchymal differentiation. The transcriptomic profiles of MBCs have been reported to vary according to the type of metaplastic elements. The somatic genetic alterations that underpin this breast cancer subtype remain to be fully characterized. Here we sought to define the genomic landscape of MBCs, whether different subtypes of MBC would be driven by distinct constellations of genetic alterations, and to investigate functionally the impact of mutations affecting WNT pathway genes using non-malignant breast epithelial cells. Methods: Thirty-five MBCs were retrieved from the pathology department of the authors' institutions and classified into the MBC histologic subtypes. All but one of the MBCs were of triple-negative phenotype. DNA was extracted from microdissected tumor-normal pairs and subjected to whole-exome sequencing. Somatic genetic alterations were identified using state-of-the-art bioinformatics algorithms. The genomic profiles of MBCs were compared to those of 69 common type TNBCs from The Cancer Genome Atlas. Overall mutation rates were compared using the Mann Whitney U test, and the frequency of mutations in each gene was compared using Fisher's exact test. RNA was extracted from a subset of MBCs and subjected to WNT signaling pathway activation analysis with the RT2 Profiler PCR Array. Triple-negative non-malignant breast epithelial cells (MCF10A and MCF12A) and cancer cell lines were utilized for 2D and 3D functional studies. Results: Whole-exome analysis revealed that MBCs displayed a median of 103 (15-344) somatic mutations, which did not differ from the median number of somatic mutations in common type TNBCs (76, range 14-233). The most frequent recurrently mutated cancer genes included TP53 (69%) and PIK3CA (29%). MBCs more frequently harbored mutations in PI3K pathway genes than common type TNBCs (57% vs 22%, P Conclusions: MBCs are significantly enriched for mutations affecting PI3K and WNT pathways, highlighting the importance of the dysregulation of the WNT pathway in MBC carcinogenesis. Moreover, our findings suggest that specific mutations are significantly associated with distinct histologic subtypes of MBCs. Citation Format: Geyer FC, Ng CK, Piscuoglio S, Wen YH, Wen H-C, Pareja F, Eberle CA, Burke KA, Lim RS, Natrajan R, Mariani O, Brogi E, Norton L, Vincent-Salomon A, Weigelt B, Reis-Filho JS. The genomic landscape of breast metaplastic carcinoma [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-05-03.

Introduction: Metaplastic breast carcinoma (MBC) is characterized by the presence of neoplastic cells displaying squamous and/or mesenchymal differentiation. Morphologic intra-tumor heterogeneity is frequent in MBCs and reported to be reflected at the transcriptomic level: whilst squamous and chondroid MBCs are preferentially of basal-like subtype, spindle cell MBCs are of claudin-low subtype. Likewise, histologically distinct components within MBCs have been shown to display distinct focal copy number alterations. Here we sought to investigate whether histologically distinct components within MBCs would be underpinned by different mutational profiles and mutational signatures. Methods: Ten MBCs with two histologically distinct components (spindle, chondroid, osseous, squamous and/ or ductal) were retrieved from the Department of Pathology of the authors' institution. The distinct components of each case and, in two cases, two regions of the same component were separately microdissected. DNA extracted from tumor samples (n=22) and matched normal tissues was subjected to whole-exome sequencing. Somatic genetic alterations were identified using state-of-the-art bioinformatics algorithms. Somatic mutations were classified as clonal (i.e., present virtually in all tumor cells) or subclonal using ABSOLUTE and FACETS. Mutational signatures were defined using non-negative matrix factorization. Results: Medians of 146 (56-290) somatic mutations and 108 non-synonymous somatic mutations (39-222) per tumor component were identified. The histologically distinct components of each case harbored identical clonal TP53 mutations. Additional recurrent mutations in cancer genes included those affecting PI3K pathway genes (PIK3CA, 2 cases; PIK3R1, 2 cases). Shared mutations between components of each case ranged from 34% to 99% of all mutations, with a median of 84%, of which 24% (12%-53%) were truncal (i.e., shared by and clonal in both components). Private mutations (i.e., found in only one component) ranged from 1% to 66%, with a median of 16%, of which 72% (0-100%) were non-synonymous and 1% (0-52%) were clonal. In two cases, the comparison of two histologically similar regions revealed less heterogeneity, with 94% (87%-100%) of shared mutations, whereas in these samples the median of private mutations was 6% (0-13%), of which 70% (0-100%) were non-synonymous and none were clonal. Private non-synonymous mutations affecting cancer genes included those in PIK3R1, MED12 and NOTCH1. The mutational signatures (e.g. aging or BRCA) were concordant between distinct components of each case; however, differences in the mutational signatures were observed between truncal somatic mutations and mutations restricted to individual components. Conclusions: MBCs display substantial genetic intra-tumor heterogeneity, which is more overt between histologically distinct components than between regions of similar histology. Our data suggest a genotypic-phenotypic correlation and corroborate the notion that distinct components within MBCs, although clonally related, may be driven by distinct somatic genetic alterations. Citation Format: Geyer FC, Burke KA, Papanastatiou AD, Macedo GS, Brogi E, Norton L, Wen YH, Weigelt B, Reis-Filho JS. Intra-tumor genetic heterogeneity and histologic heterogeneity within metaplastic breast cancers: Genotypic-phenotypic correlations [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-05-04.

Solid papillary carcinoma with reverse polarity (SPCRP) is a rare breast cancer subtype with an obscure etiology. In this study, we sought to describe its unique histopathologic features and to identify the genetic alterations that underpin SPCRP using massively parallel whole-exome and targeted sequencing. The morphologic and immunohistochemical features of SPCRP support the invasive nature of this subtype. Ten of 13 (77%) SPCRPs harbored hotspot mutations at R172 of the isocitrate dehydrogenase IDH2, of which 8 of 10 displayed concurrent pathogenic mutations affecting PIK3CA or PIK3R1. One of the IDH2 wild-type SPCRPs harbored a TET2 Q548* truncating mutation coupled with a PIK3CA H1047R hotspot mutation. Functional studies demonstrated that IDH2 and PIK3CA hotspot mutations are likely drivers of SPCRP, resulting in its reversed nuclear polarization phenotype. Our results offer a molecular definition of SPCRP as a distinct breast cancer subtype. Concurrent IDH2 and PIK3CA mutations may help diagnose SPCRP and possibly direct effective treatment. Cancer Res; 76(24); 7118–29. ©2016 AACR.

Introduction: Ductal carcinoma in situ (DCIS) is a bona fide non-obligate precursor of invasive carcinoma. Single cell sequencing studies have revealed intra-lesion genetic heterogeneity in DCIS and shown that progression to invasive ductal carcinoma (IDC) may occur through different mechanisms, including the selection of a subpopulation of tumor cells, acquisition of new genetic alterations or multi-clonal invasion. Here, we sought to investigate the genetic heterogeneity of DCIS, and to document further the clonal selection process accompanying progression to IDC. Materials and methods: Synchronous DCIS (n=16) and IDC (n=15) samples from 14 patients were microdissected separately, and DNA samples of tumor and matched normal tissues were subjected to whole-exome sequencing (WES; n=27) or massively parallel targeted sequencing of all coding regions of ≥410 cancer-related genes (n=4). Somatic genetic alterations and mutational signatures were identified using state-of-the-art bioinformatics algorithms. PyClone was employed to define the clonal architecture of each DCIS and IDC and infer the clonal shifts accompanying progression from DCIS to IDC. Results: DCIS were found to harbor recurrent somatic mutations affecting PIK3CA (50%), GATA3 (44%), TP53 (38%), CBFB (19%), PTEN (13%), and AKT1 (13%), which are genes known to be significantly mutated in invasive breast cancers. Despite the genomic similarities between matched DCIS and IDCs, NOTCH2 and MYC were found to be amplified solely in the IDC component of two cases, and PPM1D amplification was restricted to the DCIS component of another case. The mutational signature ascribed to aging (i.e. signature 1) was the predominant mutational signature in the DCIS and IDCs analyzed. PyClone analysis revealed that all synchronous DCIS and IDC studied here were clonally related and confirmed the previous observation that DCIS displays intra-lesion genetic heterogeneity. Evidence of clonal selection in the progression from DCIS to IDC was observed in three cases, whereby a minor DCIS subclone likely constituted the substrate for the development of IDC. In one of these cases, from a patient with a BRCA1 germline pathogenic mutation, we observed a shift from the mutational signature associated with defective homologous recombination DNA repair (i.e. signature 3) to the APOBEC-related mutational signatures (i.e. signatures 2 and 13) in the progression from DCIS to IDC. Conclusion: Intra-lesion genetic heterogeneity is a common feature in DCIS synchronously diagnosed with IDC. Our findings corroborate the notion that DCIS is a direct non-obligate precursor of IDC, and that clonal selection in the progression of DCIS to IDC may be present in a subset of cases, but is unlikely to constitute the most frequent mechanism of progression. Citation Format: Lee JY, Bi R, Pareja F, Geyer FC, Brown D, Wen HY, Norton L, Hicks J, Weigelt B, Reis-Filho JS. Whole exome sequencing analysis of the progression from ductal carcinoma in situ to invasive ductal carcinoma [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-01-02.

Background: The PALB2 gene encodes the partner and localizer of the BRCA2 protein, which participates in homologous recombination during DNA repair via an interaction with BRCA1 and BRCA2. Germline mutations in PALB2 are associated with an increased risk of breast cancer, with a cumulative risk of 35% by age 70 in female PALB2 mutant carriers. The aims of this project were to characterize the genomic landscape of PALB2 breast cancers and define the differences in the repertoire of somatic genetic alterations and mutational signatures between PALB2, BRCA1 and BRCA2 breast cancers. Methods: Representative samples from fourteen breast cancers from patients with known PALB2 germline mutations (seven frame-shift (2 H1170fs, 3 K346fs, 1 T841fs and 1 L531fs), five truncating (3 W1038* and 2 Q775*) and two missense (W1140G and L35P)) were microdissected to ensure a tumor cell content of >70%. DNA samples from microdissected tumors and their matched normal counterparts were subjected to whole exome sequencing on an Illumina HiSeq2000 to a median depth of 118x (range 33-193x). Somatic single nucleotide variations were detected using MuTect, and small insertions and deletions were identified using Strelka and Varscan2. Using ABSOLUTE and FACETS, we investigated the presence of loss of heterozygosity (LOH) of the PALB2 wild-type allele in these tumors. In addition, the mutational signatures and large scale state transitions (LSTs) were defined. The repertoire of somatic mutations identified in PALB2 breast cancers was compared to that of breast cancers from BRCA1 (n=11) and BRCA2 (n=10) germline mutation carriers from The Cancer Genome Atlas study. Results: PALB2 breast cancers were found to harbor a median of 80 somatic mutations (range 22-286) and one somatic mutation (range 0-13) affecting known cancer genes. Somatic loss of the PALB2 wild-type allele was found in five cases, and in three additional cases, a second PALB2 somatic mutation likely constituted the second 'hit' (two with truncating mutations, Q479* and Q61*, and one with a Q921fs frameshift mutation). Six PALB2 breast cancers displayed the BRCA mutations signature; of these, five had PALB2 bi-allelic inactivation (three LOH of the wild-type allele and two a second PALB2 somatic mutation). 71% of the samples were found to have LSTs, including all cases with a BRCA mutational signature. A significant association between PALB2 bi-allelic inactivation and concurrent BRCA signature and high LST was observed (p=0.015). Breast cancers from PALB2 mutation carriers had fewer somatic TP53 mutations than BRCA1 breast cancers (3/14, 21% vs 9/11, 82%, p=0.004), but no difference in the repertoire of somatic mutations compared to that of BRCA2 breast cancers. Conclusions: PALB2 breast cancers were found to harbor pathogenic mutations in driver genes, including TP53, PIK3CA, NF1 and NCOR1, however lacked highly recurrent somatic mutations. Unlike BRCA1/2 breast cancers, the majority of breast cancers from PALB2 germline mutation carriers lacked LOH of the PALB2 wild-type allele. Importantly, however, an association between PALB2 bi-allelic inactivation and the BRCA mutational signature and LSTs was observed, providing additional evidence for a homologous recombination-deficient phenotype at least in a subset of PALB2 cancers. Citation Format: Burke KA, Berman S, Geyer FC, Piscuoglio S, Ng CK, Wen YH, Mannermaa A, Peterlongo P, Tondini C, Janatova M, Soo Hwang T, Ng P-S, Looi LM, Chenevix-Trench G, Southey MC, Weigelt B, Foulkes W, Tischkowitz M, Reis-Filho JS, PALB2 Interest Group. Mutational landscape of breast cancers from PALB2 germline mutation carriers [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P2-03-01.

The small G-protein NRAS is mutated in 22% of human melanomas, whereas the related proteins KRAS and HRAS are mutated in only 2% and 1% of melanomas, respectively. We have developed a mouse model of melanoma in which Cre recombinase/LoxP technology is used to drive inducible expression of G12VKRAS in the melanocytic lineage. The mice develop skin hyperpigmentation, nevi, and tumors that bear many of the cardinal histopathology features and molecular characteristics of human melanoma. These tumors invade and destroy the underlying muscles and cells derived from them can grow as subcutaneous tumors and colonize the lungs of nude mice. These data establish that oncogenic KRAS can be a founder event in melanomagenesis. Cancer Res; 70(13); 5549–57. ©2010 AACR.

Adenomyoepithelioma (AME) is a rare biphasic proliferative breast lesion, which may resemble salivary gland epithelial-myoepithelial carcinomas (EMCs). Most AMEs have an indolent clinical course, but malignant transformation and local and distant recurrences have been reported. We sought to define the mutational landscape of AMEs and investigate the functional impact of recurrent likely pathogenic mutations identified in these tumors. Nineteen AMEs were subjected to whole-exome massively parallel sequencing (MPS, n=7) or targeted capture MPS using MSK-IMPACT assay (n=12). Somatic genetic alterations and the cancer cell fraction of mutations were defined using state-of-the-art bioinformatics algorithms. Selected genes (i.e. HRAS and PIK3CA) were subjected to Sanger sequencing in a series of 17 additional AMEs (total n=36). Non-tumorigenic mammary epithelial cells (i.e. MCF10A, MCF10A with the PIK3CAH1047R mutation and MCF12A), which are estrogen receptor (ER)-negative, were utilized for 2D and 3D functional studies. Of 36 cases, 22 were ER-positive and 14 were ER-negative. MPS analysis revealed a low mutation burden and HRASQ61 and PIK3CA hotspot mutations in 6/19 (32%) and 11/19 (58%) AMEs, respectively. All HRASQ61 and all but one PIK3CA mutations were clonal. ER-positive and ER-negative AMEs were fundamentally histologically and genetically distinct. Whilst ER-positive AMEs displayed recurrent PIK3CA mutations (50%, 11/22) but lacked HRAS mutations, ER-negative AMEs displayed, in addition to PIK3CA mutations (57%, 8/14), recurrent HRASQ61 mutations (57%, 8/14). HRASQ61 mutations co-occurred with PIK3CA mutations (50%, 4/8), PIK3R1 deletions (12.5%, 1/8) and/or CDKN2A homozygous deletions (25%, 2/8). HRASQ61 mutations, but not PIK3CA mutations, were significantly associated with ER-negativity (100% vs 21%), concurrent carcinoma (50% vs 7%), axillary metastases (38% vs 0%), high proliferation (63% vs 4%), necrosis (63% vs 11%) and nuclear pleomorphism (75% vs 29%). In vitro forced HRASQ61R expression in MCF10A and MCF12A cells resulted in increased proliferation and transformation. In 3D organotypic cell cultures, forced HRASQ61R resulted in a highly disorganized growth pattern, a partial loss of epithelial phenotype and acquisition of aberrant myoepithelial differentiation, which was more overt in PIK3CA-mutant MCF10A cells. In conclusion, AMEs are phenotypically and genetically heterogeneous. Whilst PIK3CA hotspot mutations occur across the spectrum of lesions, HRASQ61 hotspot mutations are restricted to ER-negative AMEs, which should arguably be classified as breast EMCs. Our genomic and functional analyses are consistent with the notion that HRASQ61 mutations are driver events in the pathogenesis of ER-negative AMEs and may be sufficient for the acquisition of myoepithelial differentiation in breast cells. Citation Format: Felipe C. Geyer, Kathleen A. Burke, Anqi Li, Anastasios D. Papanastatiou, Fresia Pareja, Anne S. Schulteis, Charlotte K. Ng, Salvatore Piscuoglio, Marcia Edelweiss, Luciano G. Martelotto, Pier Selenica, Maria R. Filippo, Gabriel S. Macedo, Achim Jungbluth, Hannah Y. Wen, Juan Palazzo, Zsuzsanna Varga, Emad Rakha, Ian O. Ellis, Brian Rubin, Britta Weigelt, Jorge S. Reis-Filho. Massively parallel sequencing analysis of breast adenomyoepitheliomas reveals the heterogeneity of the disease and identifies a subset driven by HRAS hotspot mutations [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 3379. doi:10.1158/1538-7445.AM2017-3379

Background: BRCA1 or BRCA2 germline mutations account for a substantial proportion of hereditary breast cancers. The majority of BRCA1-associated breast cancers are of triple-negative phenotype (estrogen receptor (ER)-, progesterone receptor (PR)- and HER2-negative) and harbor TP53 somatic mutations. BRCA2-associated cancers are less homogeneous and often ER-positive. Systematic analyses of the profiles of somatic genetic alterations in BRCA1- and BRCA2-breast cancers have yet to be reported. Here we sought to determine the repertoire of somatic mutations and copy number alterations (CNAs) in breast cancers occurring in patients with BRCA1 or BRCA2 germline mutations.. Methods: Eleven BRCA1- and five BRCA2-associated breast cancers were microdissected. DNA was extracted from microdissected frozen tumor-normal pairs and subjected to targeted capture massively parallel sequencing using the MSK-IMPACT platform, which includes all exons and selected introns and regulatory regions of 410 key cancer genes. Somatic single nucleotide variants, small insertions and deletions, CNAs and the cancer cell fraction (CCF) of each alteration were defined using state-of-the-art bioinformatics algorithms. Results: 82% (9/11) and 18% (2/11) of BRCA1-breast cancers were triple-negative and ER-positive/HER2-negative, respectively. All BRCA2-cancers were ER-positive, of which one was HER2-positive. Sequencing analysis revealed a median of four (2-11) and two (0-6) non-synonymous somatic mutations in BRCA1- and BRCA2-breast cancers, respectively. Within BRCA1-breast cancers all but one (10/11, 91%) harbored TP53 clonal somatic mutations, and clonal somatic loss of the BRCA1 wild-type allele was found in 8 of these cases. The BRCA1-breast cancer lacking these alterations was ER-positive and the only case harboring a PIK3CA mutation. Additional clonal mutations identified in BRCA1-breast cancers included somatic mutations affecting EGFR and RB1. Subclonal mutations in known cancer genes (e.g. GATA3 and PTEN) were also identified, suggesting intra-tumor genetic heterogeneity. All BRCA2-breast cancers analyzed displayed clonal loss of the wild-type of BRCA2, but no gene was found to be recurrently mutated. Conclusions: BRCA1- and BRCA2-breast cancers are both characterized by clonal inactivation of the BRCA1 and BRCA2 wild-type alleles, respectively, which likely constitute early genetic events. Within BRCA1-breast cancers, TP53 mutations are highly recurrent and clonal, and may precede somatic loss of the BRCA1 wild-type allele, as the latter was subclonal in one case harboring a clonal TP53 somatic mutation. Citation Format: Gabriel S. Macedo, Kathleen A. Burke, Salvatore Piscuoglio, Charlotte K. Y. Ng, Felipe C. Geyer, Luciano G. Martelotto, Anastasios D. Papanastatiou, Maria R. De Filippo, Anne M. Schultheis, Edi Brogi, Mark E. Robson, Hannah Y. Wen, Britta Weigelt, Jorge S. Reis-Filho. The landscape of somatic genetic alterations in BRCA1 and BRCA2 breast cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 100.

Background: Mucinous breast carcinoma (MBC) is a special histologic type of breast cancer, comprising approximately 2% of all invasive breast carcinomas. Morphologically, MBCs are characterized by clusters of tumor cells floating in large amounts of extracellular mucin. MBCs are preferentially of low histologic grade and estrogen receptor (ER)-positive and HER2-negative. MBCs have been shown to lack the hallmark 1q gains and 16q losses found in low-grade ER-positive invasive ductal carcinomas of no special type (IDC-NSTs). Here we sought to characterize the mutational landscape and copy number alterations (CNA) of MBCs by whole exome sequencing analysis. Material and Methods: Frozen sections from 25 pure MBCs (n = 13 mucinous A (paucicellular), n = 12 mucinous B (hypercellular)) were subjected to microdissection to ensure a tumor cell content >85%. In addition, five cases of mixed mucinous carcinomas composed of mucinous and IDC-NST components were included in this study. In these cases, the MBC and IDC-NST components were microdissected separately. DNA samples extracted from microdissected tumors and matched normal counterparts were subjected to whole exome sequencing on an Illumina HiSeq2000. Somatic single nucleotide variants (SNVs) were identified using MuTect and somatic insertions and deletions (indels) were identified using Strelka and Varscan2. Somatic CNAs were defined using FACETS. Mutational frequencies in MBCs were compared to those of luminal IDC-NSTs from The Cancer Genome Atlas. Results: A median of 26 (range 5-71) non-synonymous SNVs and indels were identified per MBC. GATA3, mutated in 28% of cases, was the only significantly mutated gene as defined by MutSigCV (q Conclusions: Our data suggest that the repertoires of somatic mutations and gene CNAs of MBCs are distinct from those of luminal IDC-NSTs, and that MBCs are an entity distinct from low-grade ER-positive IDC-NSTs not only at the morphologic but also at the genetic level. Citation Format: Salvatore Piscuoglio, Charlotte KY Ng, Felipe C. Geyer, Carey A. Eberle, Elena Guerini-Rocco, Caterina Marchio, Anne Vincent-Salomon, Jorge S. Reis-Filho, Britta Weigelt. The mutational landscape of mucinous carcinomas of the breast. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 91.

Amplification of fibroblast growth factor receptor 1 (FGFR1) occurs in ∼10% of breast cancers and is associated with poor prognosis. However, it is uncertain whether overexpression of FGFR1 is causally linked to the poor prognosis of amplified cancers. Here, we show that FGFR1 overexpression is robustly associated with FGFR1 amplification in two independent series of breast cancers. Breast cancer cell lines with FGFR1 overexpression and amplification show enhanced ligand-dependent signaling, with increased activation of the mitogen-activated protein kinase and phosphoinositide 3-kinase–AKT signaling pathways in response to FGF2, but also show basal ligand-independent signaling, and are dependent on FGFR signaling for anchorage-independent growth. FGFR1-amplified cell lines show resistance to 4-hydroxytamoxifen, which is reversed by small interfering RNA silencing of FGFR1, suggesting that FGFR1 overexpression also promotes endocrine therapy resistance. FGFR1 signaling suppresses progesterone receptor (PR) expression in vitro, and likewise, amplified cancers are frequently PR negative, identifying a potential biomarker for FGFR1 activity. Furthermore, we show that amplified cancers have a high proliferative rate assessed by Ki67 staining and that FGFR1 amplification is found in 16% to 27% of luminal B–type breast cancers. Our data suggest that amplification and overexpression of FGFR1 may be a major contributor to poor prognosis in luminal-type breast cancers, driving anchorage-independent proliferation and endocrine therapy resistance. Cancer Res; 70(5); 2085–94

Micropapillary carcinomas of the breast are a special type of breast cancer that account for up to 3% of breast cancers and which have an aggressive clinical behaviour. They are preferentially of intermediate to high histological grade and consistently express oestrogen receptor (ER). Previous genomic studies have demonstrated that micropapillary carcinomas are characterized by a constellation of genetic aberrations that is distinct from that of grade and ER-matched ductal carcinomas of the breast. Akin to sarcomas and leukaemias, it has recently been demonstrated that some special types of breast cancer (e.g. secretory and adenoid cystic carcinomas) are characterised by recurrent specific chromosomal translocations leading to the formation of oncogenic fusion genes. The aim of this study was to investigate whether micropapillary carcinomas of the breast harbour novel recurrent fusion genes. A series of six pure micropapillary carcinomas and two secretory carcinomas (positive controls harbouring the ETV6-NTRK3 translocation) were subjected to paired-end massively parallel RNA sequencing (RNA-seq). cDNA libraries were prepared according to standard mRNA prep Illumina protocols and run on the GAII sequencers (read length of each mate pair = 54bp; 2 lanes per sample). Data were aligned to the genome and transcriptome using Eland and mate-pairs supporting novel chimeras identified using previously validated pipelines. Fusion genes identified were validated using reverse transcription PCR (RT-PCR), Sanger sequencing and investigated in separate cohorts of micropapillary carcinomas (n=25) and other types of breast cancer (n=163) using fluorescence in situ hybridisation and RT-PCR. The sensitivity of the approach was validated by the analysis of the two secretory carcinomas, as the ETV6-NTRK3 reciprocal fusion gene was identified using our pipeline. Analysis of micropapillary carcinomas identified four novel intra-chromosomal fusion genes. Three of the fusions; SLC2A1-FAF1, ELMO2-RAE1, BCAS4-AURKA were present in a single tumour and spanned breakpoints of amplification. A fourth, TSPAN14-DYDC2, was a copy neutral event in a separate tumour sample. We also observed that the three fusions from a single patient were also present in the lymph node metastasis. All fusions were validated with RT-PCR, Sanger sequencing and/or FISH. One of the fusion genes was shown to be recurrent (index case and a mixed micropapillary carcinoma). Our results demonstrate that a proportion of micropapillary carcinomas of the breast harbour fusion transcripts. Further investigations are being undertaken to determine the functionality of these fusion genes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4829. doi:10.1158/1538-7445.AM2011-4829

14-3-3sigma was initially described as a tumor suppressor silenced in about 80% of breast tumors. Surprisingly however, in two independent culture models of breast cancer progression, we discovered 14-3-3sigma expression increased as cells transition towards malignancy, and that it contributed to motility and tumor invasion into surrounding tissues in vivo. Immunohistochemical analysis showed that 14-3-3sigma expression preferentially identified a subset of invasive breast carcinomas, and in independent breast cancer patient cohorts, 14-3-3sigma correlated with poor clinical outcome. We observed several lines of evidence that 14-3-3sigma regulates cytoskeletal architecture in cells, providing a molecular mechanism by which 14-3-3sigma may coordinate tumor progression and invasion. Funding is provided by a DOD BCRP Predoctoral Fellowship and a CBCRP Dissertation Award to AB, as well as DOE, NCI, NIH, and DOD grants to MJB. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5132.