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Background: Despite major technological and conceptual advancements, treatment decisions in HER2+ metastatic breast cancer (mBC) remain largely based on clinical evidence, with no established predictive biomarkers to direct treatment for individual patients. The tumour suppressor Neurofibromatosis 1 (NF1) has been implicated in endocrine resistance but its role remains incompletely characterized in mBC. NF1 is best known as a GTPase-activating protein (GAP) that attenuates RAS signalling. However, the GAP function is likely not limited to RAS, and NF1 has been involved in other GTP-dependent processes including cytoskeletal dynamics. Data mining and analysis of public mutational registries revealed NF1 mutations as particularly enriched in HER2+ mBC compared to other molecular subtypes. Methods: To investigate the biological consequences of NF1 loss, we generated NF1 KO HER2+ mBC cell lines (BT474 and SKBR3) by CRISPR-Cas9 and both 2D and 3D proliferations assays were used for drug sensitivity profiling; live-cell imaging, high-resolution confocal microscopy and an ad-hoc computational algorithm were employed to study cell fate and microtubule conformational changes. Patient data were obtained from the Northwestern University through a prospective observational study in mBC patients. Results: Screening of several compounds approved for HER2+ mBC showed that response was generally equal or reduced in NF1 KO vs WT cells. However, response to trastuzumab-emtansine (T-DM1) was significantly increased in NF1 KO cells (IC50 ~0,3 vs 1,6 μg/mL in NF1WT). This sensitization was not observed with other antibody drug conjugates (ADCs) like DS-8201 and was reproducible with maytansine alone, suggesting a pharmacologically relevant NF1 activity on microtubules. Using the FUCCI(Ca) reporter, which tracks cell cycle progression at single-cell level, we saw a more prominent G2/M phase arrest and cell death upon T-DM1 treatment in NF1 KO compared to WT cells. Notably, NF1 KO cells exhibited a higher frequency of aberrant mitotic figures (chromosome alignment defects and multipolar spindle formation) and stronger β-galactosidase activity, an established marker of senescence. Collectively, these results suggest that NF1 KO cells become particularly subject to T-DM1-triggered mitotic catastrophe. Dephosphorylation of GTP-bound tubulin is required for appropriate microtubular dynamics; so-called “GTP islands” within the inner microtubule region are prone to rapid repolymerization and are normally kept at low levels. We hypothesize that expanded GTP-tubulin islands generated by the loss of NF1 GAP activity is a major cause of microtubular instability in NF1 KO cells. Preliminary evidence in support of this model was obtained by quantification of GTP-tubulin with a specific antibody. Finally, we assessed the predictive role of NF1 as a biomarker for T-DM1 response in a cohort of 300 mBC patients with mutational data in circulating tumour DNA (Guardant 360); we identified 13 heavily pretreated patients (>4 prior lines) who received T-DM1, of which 3 had loss-of-function NF1 mutations and 10 were NF1 WT. Median progression-free survival was higher in NF1-mutated than WT patients (334 vs 80 days); given the small sample size, these results cannot yet be considered significant (p=0.14). Conclusions: These results provide preliminary mechanistic and clinical evidence supporting the use of NF1 loss to guide treatment in HER2+ mBC. As novel HER2-specific agents are being rapidly added to the therapeutic arsenal, we propose biology-driven criteria to identify patients that may benefit specifically from T-DM1. In addition, NF1 dependence for correct microtubular dynamics may be exploited by other inhibitors of microtubular polymerization in use as ADC payloads, further extending the potential usefulness of NF1 determination. Citation Format: Bruno A Duso, Elena Gavilán Dorronzoro, Giulia Tini, Maria R de Filippo, Emanuele Bonetti, Maria R Ippolito, Chiara Soriani, Paolo D'Amico, Simona Rodighiero, Giuseppe Curigliano, Stefano Santaguida, Massimo Cristofanilli, Pier Giuseppe Pelicci, Luca Mazzarella. NF1 mutations render HER2+ breast cancer highly sensitive to T-DM1 by altering microtubule dynamics [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 P5-13-04.

Adenosquamous carcinoma of the lung (ASC) is a rare subtype of non-small cell lung cancer, consisting of lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) components. ASC shows morphological characteristics of classic LUAD and LUSC but behaves more aggressively. Although ASC can serve as a model of lung cancer heterogeneity and transdifferentiation, its genomic background remains poorly understood. In this study, we sought to explore the genomic landscape of macrodissected LUAD and LUSC components of three ASC using whole exome sequencing (WES). Identified truncal mutations included the pan-cancer tumor-suppressor gene TP53 but also EGFR, BRAF, and MET, which are characteristic for LUAD but uncommon in LUSC. No truncal mutation of classical LUSC driver mutations were found. Both components showed unique driver mutations that did not overlap between the three ASC. Mutational signatures of truncal mutations differed from those of the branch mutations in their descendants LUAD and LUSC. Most common signatures were related to aging (1, 5) and smoking (4). Truncal chromosomal copy number aberrations shared by all three ASC included losses of 3p, 15q and 19p, and an amplified region in 5p. Furthermore, we detected loss of STK11 and SOX2 amplification in ASC, which has previously been shown to drive transdifferentiation from LUAD to LUSC in preclinical mouse models. Conclusively, this is the first study using WES to elucidate the clonal evolution of ASC. It provides strong evidence that the LUAD and LUSC components of ASC share a common origin and that the LUAD component appears to transform to LUSC.

Hirschsprung (HSCR) Associated Enterocolitis (HAEC) is a common life-threatening complication in HSCR. HAEC is suggested to be due to a loss of gut homeostasis caused by impairment of immune system, barrier defense, and microbiome, likely related to genetic causes. No gene has been claimed to contribute to HAEC occurrence, yet. Genetic investigation of HAEC by Whole-Exome Sequencing (WES) on 24 HSCR patients affected (HAEC) or not affected (HSCR-only) by enterocolitis and replication of results on a larger panel of patients allowed the identification of the HAEC susceptibility variant p.H187Q in the Oncostatin-M receptor (OSMR) gene (14.6% in HAEC and 5.1% in HSCR-only, p = 0.0024). Proteomic analysis on the lymphoblastoid cell lines from one HAEC patient homozygote for this variant and one HAEC patient not carrying the variant revealed two well distinct clusters of proteins significantly up or downregulated upon OSM stimulation. A marked enrichment in immune response pathways (q &lt, 0.0001) was shown in the HAEC H187 cell line, while proteins upregulated in the HAEC Q187 lymphoblasts sustained pathways likely involved in pathogen infection and inflammation. In conclusion, OSMR p.H187Q is an HAEC susceptibility variant and perturbates the downstream signaling cascade necessary for the gut immune response and homeostasis maintenance.

Therapy resistance and metastatic processes in prostate cancer (PCa) remain undefined, due to lack of experimental models that mimic different disease stages. We describe a novel androgen-dependent PCa patient-derived xenograft (PDX) model from treatment-naïve, soft tissue metastasis (PNPCa). RNA and whole-exome sequencing of the PDX tissue and organoids confirmed transcriptomic and genomic similarity to primary tumor. PNPCa harboursBRCA2 and CHD1somatic mutations, shows anSPOP/FOXA1-like transcriptomic signature and microsatellite instability, which occurs in 3% of advanced PCa and has never been modelledin vivo. Comparison of the treatment-naïve PNPCa with additional metastatic PDXs (BM18, LAPC9), in a medium-throughput organoid screen of FDA-approved compounds, revealed differential drug sensitivities. Multikinase inhibitors (ponatinib, sunitinib, sorafenib) were broadly effective on all PDX- and patient-derived organoids from advanced cases with acquired resistance to standard-of-care compounds. This proof-of-principle study may provide a preclinical tool to screen drug responses to standard-of-care and newly identified, repurposed compounds.

Therapy resistance and metastatic processes in prostate cancer (PCa) remain undefined, due to lack of experimental models that mimic different disease stages. We describe an androgen-dependent PCa patient-derived xenograft (PDX) model from treatment-naïve, soft tissue metastasis (PNPCa). RNA and whole-exome sequencing of the PDX tissue and organoids confirmed transcriptomic and genomic similarity to primary tumor. PNPCa harbors BRCA2 and CHD1 somatic mutations, shows an SPOP/FOXA1-like transcriptomic signature and microsatellite instability, which occurs in 3% of advanced PCa and has never been modeled in vivo. Comparison of the treatment-naïve PNPCa with additional metastatic PDXs (BM18, LAPC9), in a medium-throughput organoid screen of FDA-approved compounds, revealed differential drug sensitivities. Multikinase inhibitors (ponatinib, sunitinib, sorafenib) were broadly effective on all PDX- and patient-derived organoids from advanced cases with acquired resistance to standard-of-care compounds. This proof-of-principle study may provide a preclinical tool to screen drug responses to standard-of-care and newly identified, repurposed compounds., To date, patients still succumb to cancer, due to tumors not responding to therapy or ultimately acquiring resistance. Here the authors show that by exploiting patient derived organoids and a treatment-naïve patient derived xenograft, patient therapy can be personalized.

Simple Summary Currently, there is a need for prognostic tools that can stratify patients, who present with primary disease, based on whether they are at low or high risk for drug resistant and hormone-independent lethal metastatic prostate cancer. The aim of our study was to assess the potentially added value of tumor microenvironment (stroma) components for the characterisation of prostate cancer. By utilising patient derived-xenograft models we show that the molecular properties of the stroma cells are highly responsive to androgen hormone levels, and considerable ECM remodelling processes take place not only in androgen-dependent but also in androgen-independent tumor models. Transcriptomic mechanisms linked to osteotropism are conserved in bone metastatic xenografts, even when implanted in a different microenvironment. A stroma-specific gene list signature was identified, which highly correlates with Gleason score, metastasis progression and progression-free survival, and thus could potentially complement current patient stratification methods. Abstract Resistance acquisition to androgen deprivation treatment and metastasis progression are a major clinical issue associated with prostate cancer (PCa). The role of stroma during disease progression is insufficiently defined. Using transcriptomic and proteomic analyses on differentially aggressive patient-derived xenografts (PDXs), we investigated whether PCa tumors predispose their microenvironment (stroma) to a metastatic gene expression pattern. RNA sequencing was performed on the PCa PDXs BM18 (castration-sensitive) and LAPC9 (castration-resistant), representing different disease stages. Using organism-specific reference databases, the human-specific transcriptome (tumor) was identified and separated from the mouse-specific transcriptome (stroma). To identify proteomic changes in the tumor (human) versus the stroma (mouse), we performed human/mouse cell separation and subjected protein lysates to quantitative Tandem Mass Tag labeling and mass spectrometry. Tenascin C (TNC) was among the most abundant stromal genes, modulated by androgen levels in vivo and highly expressed in castration-resistant LAPC9 PDX. The tissue microarray of primary PCa samples (n = 210) showed that TNC is a negative prognostic marker of the clinical progression to recurrence or metastasis. Stroma markers of osteoblastic PCa bone metastases seven-up signature were induced in the stroma by the host organism in metastatic xenografts, indicating conserved mechanisms of tumor cells to induce a stromal premetastatic signature. A 50-gene list stroma signature was identified based on androgen-dependent responses, which shows a linear association with the Gleason score, metastasis progression and progression-free survival. Our data show that metastatic PCa PDXs, which differ in androgen sensitivity, trigger differential stroma responses, which show the metastasis risk stratification and prognostic biomarker potential.

Neuroendocrine breast carcinomas (NBCs) account for 2-5% of all invasive breast cancers, and are histologically similar to neuroendocrine tumours from other sites. They typically express oestrogen receptor (ER), and are HER2-negative and of luminal 'intrinsic' subtype. Here, we sought to define the mutational profile of NBCs, and to investigate whether NBCs and common forms of luminal (ER+ /HER2- ) breast carcinoma show distinct repertoires of somatic mutations. Eighteen ER+ /HER2- NBCs, defined as harbouring >50% of tumour cells expressing chromogranin A and/or synaptophysin, and matched normal tissues were microdissected and subjected to massively parallel sequencing targeting all exons of 254 genes most frequently mutated in breast carcinomas and/or related to DNA repair. Their mutational repertoire was compared with that of ER+ /HER2- breast carcinomas (n = 240), PAM50-defined luminal breast carcinomas (luminal A, n = 209; luminal B, n = 111) and invasive lobular carcinomas (n = 127) from The Cancer Genome Atlas. NBCs were found to harbour a median of 4.5 (range 1-11) somatic mutations, similar to that of luminal B breast carcinomas (median = 3, range 0-17) but significantly higher than that of luminal A breast carcinomas (median = 3, range 0-18, p = 0.02). The most frequently mutated genes were GATA3, FOXA1, TBX3, and ARID1A (3/18, 17%), and PIK3CA, AKT1, and CDH1 (2/18, 11%). NBCs less frequently harboured PIK3CA mutations than common forms of ER+ /HER2- , luminal A and invasive lobular carcinomas (p

International audience; An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

International audience; Metastasis is the main cause of death for patients with breast cancer. Many studies have characterized the genomic landscape of breast cancer during its early stages. However, there is evidence that genomic alterations are acquired during the evolution of cancers from their early to late stages, and that the genomic landscape of early cancers is not representative of that of lethal cancers1-7. Here we investigated the landscape of somatic alterations in 617 metastatic breast cancers. Nine driver genes (TP53, ESR1, GATA3, KMT2C, NCOR1, AKT1, NF1, RIC8A and RB1) were more frequently mutated in metastatic breast cancers that expressed hormone receptors (oestrogen and/or progesterone receptors; HR+) but did not have high levels of HER2 (HER2-; n = 381), when compared to early breast cancers from The Cancer Genome Atlas. In addition, 18 amplicons were more frequently observed in HR+/HER2- metastatic breast cancers. These cancers showed an increase in mutational signatures S2, S3, S10, S13 and S17. Among the gene alterations that were enriched in HR+/HER2- metastatic breast cancers, mutations in TP53, RB1 and NF1, together with S10, S13 and S17, were associated with poor outcome. Metastatic triple-negative breast cancers showed an increase in the frequency of somatic biallelic loss-of-function mutations in genes related to homologous recombination DNA repair, compared to early triple-negative breast cancers (7% versus 2%). Finally, metastatic breast cancers showed an increase in mutational burden and clonal diversity compared to early breast cancers. Thus, the genomic landscape of metastatic breast cancer is enriched in clinically relevant genomic alterations and is more complex than that of early breast cancer. The identification of genomic alterations associated with poor outcome will allow earlier and better selection of patients who require the use of treatments that are still in clinical trials. The genetic complexity observed in advanced breast cancer suggests that such treatments should be introduced as early as possible in the disease course.

Adenoid cystic carcinoma of the breast is a rare histological type of triple-negative breast cancer with an indolent clinical behavior, often driven by the MYB-NFIB fusion gene. Here we sought to define the repertoire of somatic genetic alterations in two adenoid cystic carcinomas associated with high-grade triple-negative breast cancer. The different components of each case were subjected to copy number profiling and massively parallel sequencing targeting all exons and selected regulatory and intronic regions of 488 genes. Reverse transcription PCR and fluorescence in situ hybridization were employed to investigate the presence of the MYB-NFIB translocation. The MYB-NFIB fusion gene was detected in both adenoid cystic carcinomas and their associated high-grade triple-negative breast cancer components. Although the distinct components of both cases displayed similar patterns of gene copy number alterations, massively parallel sequencing analysis revealed intratumor genetic heterogeneity. In case 1, progression from the trabecular adenoid cystic carcinoma to the high-grade triple-negative breast cancer was found to involve clonal shifts with enrichment of mutations affecting EP300, NOTCH1, ERBB2 and FGFR1 in the high-grade triple-negative breast cancer. In case 2, a clonal KMT2C mutation was present in the cribriform adenoid cystic carcinoma, solid adenoid cystic carcinoma and high-grade triple-negative breast cancer components, whereas a mutation affecting MYB was present only in the solid and high-grade triple-negative breast cancer areas and additional three mutations targeting STAG2, KDM6A and CDK12 were restricted to the high-grade triple-negative breast cancer. In conclusion, adenoid cystic carcinomas of the breast with high-grade transformation are underpinned by the MYB-NFIB fusion gene and, akin to other forms of cancer, may be constituted by a mosaic of cancer cell clones at diagnosis. The progression from adenoid cystic carcinoma to high-grade triple-negative breast cancer of no special type may involve the selection of neoplastic clones and/or the acquisition of additional genetic alterations.

Microglandular adenosis (MGA) is a rare proliferative lesion of the breast composed of small glands lacking myoepithelial cells and lined by S100-positive, oestrogen receptor (ER)-negative, progesterone receptor (PR)-negative, and HER2-negative epithelial cells. There is evidence to suggest that MGA may constitute a non-obligate precursor of triple-negative breast cancer (TNBC). We sought to define the genomic landscape of pure MGA and of MGA, atypical MGA (AMGA) and associated TNBCs, and to determine whether synchronous MGA, AMGA, and TNBCs would be clonally related. Two pure MGAs and eight cases of MGA and/or AMGA associated with in situ or invasive TNBC were collected, microdissected, and subjected to massively parallel sequencing targeting all coding regions of 236 genes recurrently mutated in breast cancer or related to DNA repair. Pure MGAs lacked clonal non-synonymous somatic mutations and displayed limited copy number alterations (CNAs); conversely, all MGAs (n = 7) and AMGAs (n = 3) associated with TNBC harboured at least one somatic non-synonymous mutation (range 3-14 and 1-10, respectively). In all cases where TNBCs were analyzed, identical TP53 mutations and similar patterns of gene CNAs were found in the MGA and/or AMGA and in the associated TNBC. In the MGA/AMGA associated with TNBC lacking TP53 mutations, somatic mutations affecting PI3K pathway-related genes (eg PTEN, PIK3CA, and INPP4B) and tyrosine kinase receptor signalling-related genes (eg ERBB3 and FGFR2) were identified. At diagnosis, MGAs associated with TNBC were found to display subclonal populations, and clonal shifts in the progression from MGA to AMGA and/or to TNBC were observed. Our results demonstrate the heterogeneity of MGAs, and that MGAs associated with TNBC, but not necessarily pure MGAs, are genetically advanced, clonal, and neoplastic lesions harbouring recurrent mutations in TP53 and/or other cancer genes, supporting the notion that a subset of MGAs and AMGAs may constitute non-obligate precursors of TNBCs.

Uterine adenosarcomas (UAs) are biphasic lesions composed of a malignant mesenchymal (ie stromal) component and an epithelial component. UAs are generally low-grade and have a favourable prognosis, but may display sarcomatous overgrowth (SO), which is associated with a worse outcome. We hypothesized that, akin to breast fibroepithelial lesions, UAs are mesenchymal neoplasms in which clonal somatic genetic alterations are restricted to the mesenchymal component. To characterize the somatic genetic alterations in UAs and to test this hypothesis, we subjected 20 UAs to a combination of whole-exome (n = 6), targeted capture (n = 13) massively parallel sequencing (MPS) and/or RNA sequencing (n = 6). Only three genes, FGFR2, KMT2C and DICER1, were recurrently mutated, all in 2/19 cases; however, 26% (5/19) and 21% (4/19) of UAs harboured MDM2/CDK4/HMGA2 and TERT gene amplification, respectively, and two cases harboured fusion genes involving NCOA family members. Using a combination of laser-capture microdissection and in situ techniques, we demonstrated that the somatic genetic alterations detected by MPS were restricted to the mesenchymal component. Furthermore, mitochondrial DNA sequencing of microdissected samples revealed that epithelial and mesenchymal components of UAs were clonally unrelated. In conclusion, here we provide evidence that UAs are genetically heterogeneous lesions and mesenchymal neoplasms.

Therapy resistance and metastatic processes in prostate cancer (PCa) remain undefined, mainly due to the lack of experimental models representing different disease stages. We aim to provide functional experimental and preclinical drug tools applicable for direct use with patient-derived material. Biopsies from metastatic PCa were used for the establishment of patient-derived xenografts (PDXs) by subcutaneous implantation. In vivo tumor growth kinetic in response to androgens was assessed by surgical castration and testosterone supplementation. RNA and whole-exome sequencing (WES) and organoid drug screens were used to characterize the genomic, transcriptomic, and functional properties. Organoid culture methodology was adapted in order to set up an automated medium-throughput organoid drug screen (Nexus Theragnostics automated platform) for screening standard-of-care compounds and candidates for drug repurposing. A novel case of PCa xenograft model derived from soft tissue metastasis (PNPCa) was established. RNAseq and WES confirmed transcriptomic and genomic similarity to the primary tumor. PNPCa harbors BRAC2 and CHD1 mutations and shows SPOP-like and FOXA1-like transcriptomic signature with microsatellite instability. PNPCa tumor growth is inhibited after androgen deprivation by castration, while androgen replacement leads to tumor reformation. No spontaneous tumor growth occurred after prolonged period of castration; however, scarce micrometastases were found in the bone marrow and lymph nodes. The treatment-naive and androgen-dependent properties of the PNPCa made it a candidate model for identification of potent drug compounds. PNPCa-derived organoid screens on standard-of-care and 74 FDA-approved compounds showed that mTOR pathway and multi-tyrosine kinase inhibitors, used for clinical treatment of other cancer types, have high impact on PCa organoid viability. Application of the organoid drug screen panel on two additional metastatic PDXs of advanced disease has allowed shortlisting of compounds for repurposing use in patient-derived organoid screens that can be routinely performed in a timeframe of few weeks, and thus, provide information on treatment decision. Treatment response of a treatment-naive, early metastatic PCa case (PNPCa PDX) highlights the potential of organoid screens to assess therapy response with extended applications for personalized screens. Citation Format: Sofia Karkampouna, Federico la Manna, Maria R. De Filippo, Mirjam Kiener, Marta De Menna, Eugenio Zoni, Joel Grosjean, Irena Klima, Andrea Garofoli, Marco Bolis, Jean-Philippe Theurillat, Vera Genitsch, David Keller, Booij Tijmen, Christian U. Stirnimann, Andrea Sboner, Charlotte K.Y. Ng, Salvatore Piscuoglio, Martin Spahn, Mark A. Rubin, George N. Thalmann, Marianna Kruithof-de Julio. Patient-derived xenograft and organoids models of prostate cancer [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr B18.

// Leticia De Mattos-Arruda 1, 2, 3, * , Charlotte K. Y. Ng 1, 4, 5, * , Salvatore Piscuoglio 1, 4 , Maria Gonzalez-Cao 6 , Raymond S. Lim 1 , Maria R. De Filippo 1 , Nicola Fusco 1 , Anne M. Schultheis 1 , Carolina Ortiz 2 , Santiago Viteri 6 , Alexandra Arias 2 , Gabriel S. Macedo 1 , Mafalda Oliveira 2 , Patricia Gomez 2 , Cristina Teixido 6 , Paolo Nuciforo 2 , Vicente Peg 7 , Cristina Saura 2 , Santiago Ramon y Cajal 7 , Francesc Tresserra Casas 6 , Britta Weigelt 1 , Javier Cortes 2, 3, 8 , Joan Seoane 2, 3, 9 and Jorge S. Reis-Filho 1, 10 1 Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, NY, USA 2 Vall d'Hebron Institute of Oncology (VHIO), Vall d'Hebron University Hospital, Barcelona, Spain 3 Universitat Autonoma de Barcelona, Barcelona, Spain 4 Institute of Pathology, University Hospital Basel, Basel, Switzerland 5 Department of Biomedicine, University of Basel, Basel, Switzerland 6 Quiron Dexeus University Hospital, Barcelona, Spain 7 Vall d'Hebron Institute of Research, Vall d'Hebron University Hospital, Barcelona, Spain 8 Ramon y Cajal University Hospital, Madrid, Spain 9 Institucio Catalana de Recerca i Estudis Avancats (ICREA), Barcelona, Spain 10 Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA * These authors contributed equally to this work Correspondence to: Leticia De Mattos-Arruda, email: ldmattos@vhio.net Jorge S. Reis-Filho, email: reisfilj@mskcc.org Keywords: metastatic breast cancer; HER2-positive; brain metastasis; actionable genetic alterations; personalized medicine Received: January 16, 2018 Accepted: March 12, 2018 Published: April 17, 2018 ABSTRACT Brain metastases constitute a challenge in the management of patients with HER2-positive breast cancer treated with anti-HER2 systemic therapies. Here we sought to define the repertoire of mutations private to or enriched for in HER2-positive brain metastases. Massively parallel sequencing targeting all exons of 254 genes frequently mutated in breast cancers and/or related to DNA repair was used to characterize the spatial and temporal heterogeneity of HER2-positive breast cancers and their brain metastases in six patients. Data were analyzed with state-of-the-art bioinformatics algorithms and selected mutations were validated with orthogonal methods. Spatial and temporal inter-lesion genetic heterogeneity was observed in the HER2-positive brain metastases from an index patient subjected to a rapid autopsy. Genetic alterations restricted to the brain metastases included mutations in cancer genes FGFR2, PIK3CA and ATR , homozygous deletion in CDKN2A and amplification in KRAS . Shifts in clonal composition and the acquisition of additional mutations in the progression from primary HER2-positive breast cancer to brain metastases following anti-HER2 therapy were investigated in additional five patients. Likely pathogenic mutations private to or enriched in the brain lesions affected cancer and clinically actionable genes, including ATR, BRAF, FGFR2, MAP2K4, PIK3CA, RAF1 and TP53 . Changes in clonal composition and the acquisition of additional mutations in brain metastases may affect potentially actionable genes in HER2-positive breast cancers. Our observations have potential clinical implications, given that treatment decisions for patients with brain metastatic disease are still mainly based on biomarkers assessed in the primary tumor.

Acinic cell carcinoma (ACC) of the breast is a rare form of triple-negative (that is, oestrogen receptor-negative, progesterone receptor-negative, HER2-negative) salivary gland-type tumour displaying serous acinar differentiation. Despite its triple-negative phenotype, breast ACCs are reported to have an indolent clinical behaviour. Here, we sought to define whether ACCs have a mutational repertoire distinct from that of other triple-negative breast cancers (TNBCs). DNA was extracted from microdissected formalin-fixed, paraffin-embedded sections of tumour and normal tissue from two pure and six mixed breast ACCs. Each tumour component of the mixed cases was microdissected separately. Tumour and normal samples were subjected to targeted capture massively parallel sequencing targeting all exons of 254 genes, including genes most frequently mutated in breast cancer and related to DNA repair. Selected somatic mutations were validated by targeted amplicon resequencing and Sanger sequencing. Akin to other forms of TNBC, the most frequently mutated gene found in breast ACCs was TP53 (one pure and six mixed cases). Additional somatic mutations affecting breast cancer-related genes found in ACCs included PIK3CA, MTOR, CTNNB1, BRCA1, ERBB4, ERBB3, INPP4B, and FGFR2. Copy number alteration analysis revealed complex patterns of gains and losses similar to those of common forms of TNBCs. Of the mixed cases analysed, identical somatic mutations were found in the acinic and the high-grade non-acinic components in two out of four cases analysed, providing evidence of their clonal relatedness. In conclusion, breast ACCs display the hallmark somatic genetic alterations found in high-grade forms of TNBC, including complex patterns of gene copy number alterations and recurrent TP53 mutations. Furthermore, we provide circumstantial genetic evidence to suggest that ACCs may constitute the substrate for the development of more aggressive forms of triple-negative disease.

Adenoid cystic carcinoma (AdCC) is a rare type of triple-negative breast cancer (TNBC) characterized by the presence of the MYB-NFIB fusion gene. The molecular underpinning of breast AdCCs other than the MYB-NFIB fusion gene remains largely unexplored. Here we sought to define the repertoire of somatic genetic alterations of breast AdCCs. We performed whole-exome sequencing, followed by orthogonal validation, of 12 breast AdCCs to determine the landscape of somatic mutations and gene copy number alterations. Fluorescence in situ hybridization and reverse-transcription PCR were used to define the presence of MYB gene rearrangements and MYB-NFIB chimeric transcripts. Unlike common forms of TNBC, we found that AdCCs have a low mutation rate (0.27 non-silent mutations/Mb), lack mutations in TP53 and PIK3CA and display a heterogeneous constellation of known cancer genes affected by somatic mutations, including MYB, BRAF, FBXW7, SMARCA5, SF3B1 and FGFR2. MYB and TLN2 were affected by somatic mutations in two cases each. Akin to salivary gland AdCCs, breast AdCCs were found to harbour mutations targeting chromatin remodelling, cell adhesion, RNA biology, ubiquitination and canonical signalling pathway genes. We observed that, although breast AdCCs had rather simple genomes, they likely display intra-tumour genetic heterogeneity at diagnosis. Taken together, these findings demonstrate that the mutational burden and mutational repertoire of breast AdCCs are more similar to those of salivary gland AdCCs than to those of other types of TNBCs, emphasizing the importance of histological subtyping of TNBCs. Furthermore, our data provide direct evidence that AdCCs harbour a distinctive mutational landscape and genomic structure, irrespective of the disease site of origin.

Objective The vast majority of endometrioid endometrial carcinomas (EECs) harbor mutations in the PI3K pathway. Here we sought to determine whether the type and pattern of mutations targeting different components of the PI3K pathway are distinct between microsatellite stable (MSS) and high-level microsatellite instable (MSI-H) EECs. Methods Whole exome massively parallel sequencing-based mutation data from EECs of The Cancer Genome Atlas (TCGA) were used to define the number, type and pattern of mutations affecting PI3K pathway-related genes, including AKT1 , INPP4B , MTOR , PIK3CA , PIK3R1 and PTEN . EECs were classified as MSI-H (n=70) and MSS (n=109) based on seven MSI markers assessed by TCGA. Ultramutated cases were excluded. Results Although the mutation rates and mutational signatures of MSS and MSI-H EECs were distinct, the prevalence of PI3K pathway mutations was similar between these two groups (all p >0.05), with the exception of PTEN mutations, which were more prevalent in MSI-H (61/70; 87%) than in MSS EECs (78/109; 72%; p =0.017). The PIK3CA hotspot mutations E542K, E545K, and H1047R were found to be significantly more prevalent in PIK3CA -mutant MSS (21/58, 36%) compared to PIK3CA -mutant MSI-H EECs (5/37, 13.5%; p =0.019). Conclusion Although the prevalence of mutations targeting PI3K pathway genes is similar between MSS and MSI-H EECs, PIK3CA hotspot mutations, which result in constitutive kinase activation, are significantly more prevalent in MSS than in MSI-H EECs. Our findings warrant further investigation of the role of different types of PIK3CA mutations and their predictive impact on distinct subtypes of EECs.

• A new mutation in STXBP1 gene in a patient with a clinical history of Ohtahara syndrome and a severe adverse reaction to phenytoin, co-occurring with compound heterozygous mutations in CYP2C9 gene.

Estrogen receptor β (ERβ) is a member of the nuclear receptor family of homeostatic regulators that is frequently lost in breast cancer (BC), where its presence correlates with a better prognosis and a less aggressive clinical outcome of the disease. In contrast to ERα, its closest homolog, ERβ shows significant estrogen-independent activities, including the ability to inhibit cell cycle progression and regulate gene transcription in the absence of the ligand. Investigating the nature and extent of this constitutive activity of ERβ in BC MCF-7 and ZR-75.1 cells by means of microRNA (miRNA) sequencing, we identified 30 miRNAs differentially expressed in ERβ+ versus ERβ− cells in the absence of ligand, including up-regulated oncosuppressor miRs such miR-30a. In addition, a significant fraction of >1,600 unique proteins identified in MCF-7 cells by iTRAQ quantitative proteomics were either increased or decreased by ERβ, revealing regulation of multiple cell pathways by ligand-free receptors. Transcriptome analysis showed that for a large number of proteins regulated by ERβ, the corresponding mRNAs are unaffected, including a large number of putative targets of ERβ-regulated miRNAs, indicating a central role of miRNAs in mediating BC cell proteome regulation by ERβ. Expression of a mimic of miR-30a-5p, a direct target and downstream effector of ERβ in BC, led to the identification of several target transcripts of this miRNA, including 11 encoding proteins whose intracellular concentration was significantly affected by unliganded receptor. These results demonstrate a significant effect of ligand-free ERβ on BC cell functions via modulation of the cell proteome and suggest that miRNA regulation might represent a key event in the control of the biological and clinical phenotype of hormone-responsive BC by this nuclear receptor.

Sequencing of the KCNT1, PLCB1, SCN1A and TBC1D24 loci was performed in six children with typical features of malignant migrating partial seizures of infancy (MMPSI), to verify the presence of potential disease-causing mutations, including those already reported to be associated with the disease. Sanger sequencing failed to identify in these genes the previously reported pathogenic mutations in these patients, while a comprehensive mutational scanning analysis of these four loci by targeted re-sequencing led to detection of both intronic and exonic new variants. Based on the current knowledge, the sequence variants identified here do not allow to predict functional phenotypes that might explain, at least in part, MMPSI symptoms.

Background:All carcinomas contain a variable proportion of benign stromal and immune cells, limiting the sensitivity in the identification of somatic genetic alterations. The average tumor purity of squamous cell carcinomas (SCC) of the lung is only around 50%. Low tumor content in tumor samples represents a challenge in studying intratumoral genomic heterogeneity in cancer research. Here, we applied flow-sorting to enrich for tumor cell nuclei to investigate the clonal relationship of primary SCC and matched metastases. Methods:Tumor tissues from 16 patients with primary SCC of the lung and matched metastases were used. We implemented a flow-sorting based approach to enrich for tumor nuclei as followed: after extraction of nuclei from snap-frozen or FFPE tissue, they were flow-sorted by DNA content (ploidy) and cytokeratin expression of the adherent cytoplasm, using a pan-cytokeratin (pCK) antibody. Isolated diploid and aneuploid pCK-positive tumor cell populations were subjected to whole exome sequencing (WES). DNA from diploid pCK-negative populations was used as germline control. Mutational profile and copy number aberrations (CNA) were determined to infer the clonal relationship and evolution between the primary tumors and their metastases. Results:Our flow-sorting based approach was able to enrich tumor content from 20%-50% to more than 80%-90% and to distinguish between aneuploid and diploid tumor cell populations from bulk tumor tissues. It enabled the identification of somatic mutations and CNA in both, aneuploid and diploid tumor cell populations, including potential subclonal driver mutations in ARID1A and KDM6A at low allelic frequencies. Shared and private mutations were observed in matched longitudinal tumor samples of individual patients and in synchronous distant metastases. Ploidy did not change significantly between primary tumors and relapse or distant metastases. Conclusion:We present a flow-sorting based method to enrich for tumor cell nuclei to facilitate genomic analysis, which also enabled separate analysis of aneuploid and diploid tumor populations. Our results show that the enrichment approach can be used to decode the clonal evolutionary relationship between primary tumors and their matched metastases, even in samples with low tumor cell content. Citation Format: Arthur Krause, Maria R. De Filippo, Thomas Lorber, Tanja Dietsche, Valeria Perrina, Christian Ruiz, Michael T. Barrett, Salvatore Piscuoglio, Charlotte K. Ng, Lukas Bubendorf. Enriching tumor purity using a unique flow-sorting approach to elucidate clonal evolution in matched samples of squamous cell carcinoma of the lung [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 4699.

Background: Adenosquamous carcinoma (ASC) is a rare subtype of non-small-cell lung cancer, consisting of an adenocarcinoma (AC) and a squamous cell carcinoma (SCC) component. Little is known about the molecular makeup of the two components in ASC. In this study, we applied a comprehensive analysis including whole exome sequencing (WES) and RNA sequencing to understand the evolutionary relationship between the AC and SCC components of an ASC of the lung. Methods:FFPE tumor tissue of three ASC patients was used in this study. Samples were stained with FastRed to visualize the nuclei. AC and SCC components were scratched separately from histological slides for DNA and RNA extraction. WES analysis was performed to assess the mutational profile and copy number alterations (CNAs). The transcriptome was investigated in each of the components using RNA sequencing. Results:WES analysis of the AC and SCC components of one patient revealed a common trunk of 118 mutations, including driver mutations EGFR exon 19 deletion and TP53 p.192*, indicating a common clonal origin of the components and the presence of bona fide drivers in the common ancestor. AC shows more private mutations than SCC (234 vs 134). Potential drivers are private to AC included TP53 and AKT1 mutations, while TLX1 and TRBV5-1 mutations are restricted to SCC. AC and SCC have highly similar CNA profiles, suggesting that all CNAs are early evolutionary events of the ASC. Mutational signature of the truncal mutations is similar to that of the mutations private to AC, while mutations private to SCC showed a distinct pattern enriched in T>G, suggesting that SCC may have derived from an AC ancestor cell. Transcriptomic profiling shows that genes related to oxidative phosphorylation, fatty acid metabolism and peroxisome pathways are highly expressed in AC, in contrast to genes related to epithelial-mesenchymal-transition, hedgehog signaling and IL6-JAK-STAT3-signaling pathways in SCC. Conclusion:Macroscopic dissection and separate analysis of the histologically distinct components of an ASC of the lung demonstrated the clonal relatedness of the AC and SCC. The diverse phenotype of the components is associated with distinct genetic profiles and mutational signatures. Analysis of additional ASCs of the lung may reveal genetic and/or transcriptomic underpinnings of the phenotypes. Citation Format: Arthur Krause, Maria R. De Filippo, Thomas Lorber, Spasenija Savic, Salvatore Piscuoglio, Charlotte K. Ng, Lukas Bubendorf. Comprehensive analysis of the histologically distinct components of an adenosquamous carcinoma of the lung [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 2515.

The IBTK gene encodes the major protein isoform IBTKα that was recently characterized as substrate receptor of Cul3-dependent E3 ligase, regulating ubiquitination coupled to proteasomal degradation of Pdcd4, an inhibitor of translation. Due to the presence of Ankyrin-BTB-RCC1 domains that mediate several protein-protein interactions, IBTKα could exert expanded regulatory roles, including interaction with transcription regulators. To verify the effects of IBTKα on gene expression, we analyzed HeLa and K562 cell transcriptomes by RNA-Sequencing before and after IBTK knock-down by shRNA transduction. In HeLa cells, 1285 (2.03%) of 63,128 mapped transcripts were differentially expressed in IBTK-shRNA-transduced cells, as compared to cells treated with control-shRNA, with 587 upregulated (45.7%) and 698 downregulated (54.3%) RNAs. In K562 cells, 1959 (3.1%) of 63128 mapped RNAs were differentially expressed in IBTK-shRNA-transduced cells, including 1053 upregulated (53.7%) and 906 downregulated (46.3%). Only 137 transcripts (0.22%) were commonly deregulated by IBTK silencing in both HeLa and K562 cells, indicating that most IBTKα effects on gene expression are cell type-specific. Based on gene ontology classification, the genes responsive to IBTK are involved in different biological processes, including in particular chromatin and nucleosomal organization, gene expression regulation, and cellular traffic and migration. In addition, IBTK RNA interference affected RNA maturation in both cell lines, as shown by the evidence of alternative 3′- and 5′-splicing, mutually exclusive exons, retained introns, and skipped exons. Altogether, these results indicate that IBTK differently modulates gene expression and RNA splicing in HeLa and K562 cells, demonstrating a novel biological role of this protein.

Endometrial carcinomas (ECs) are heterogeneous at the genetic level. Although TP53 mutations are highly recurrent in serous endometrial carcinomas (SECs), these are also present in a subset of endometrioid endometrial carcinomas (EECs). Here, we sought to define the frequency, pattern, distribution, and type of TP53 somatic mutations in ECs by performing a reanalysis of the publicly available data from The Cancer Genome Atlas (TCGA). A total of 228 EECs (n=186) and SECs (n=42) from the TCGA data set, for which an integrated genomic characterization was performed, were interrogated for the presence and type of TP53 mutations, and for mutations in genes frequently mutated in ECs. TP53 mutations were found in 15% of EECs and 88% of SECs, and in 91% of copy-number-high and 35% of polymerase (DNA directed), epsilon, catalytic subunit (POLE) integrative genomic subtypes. In addition to differences in prevalence, variations in the type and pattern of TP53 mutations were observed between histologic types and between integrative genomic subtypes. TP53 hotspot mutations were significantly more frequently found in SECs (46%) than in EECs (15%). TP53-mutant EECs significantly more frequently harbored a co-occurring PTEN mutation than TP53-mutant SECs. Finally, a subset of TP53-mutant ECs (22%) was found to harbor frameshift or nonsense mutations. Given that nonsense and frameshift TP53 mutations result in distinct p53 immunohistochemical results that require careful interpretation, and that EECs and SECs display different patterns, types, and distributions of TP53 mutations, the use of the TP53/p53 status alone for the differential diagnosis of EECs and SECs may not be sufficient.

Microglandular adenosis (MGA) is a rare proliferative lesion of the breast composed of small glands lacking myoepithelial cells and lined by S100-positive, oestrogen receptor (ER)-negative, progesterone receptor (PR)-negative, and HER2-negative epithelial cells. There is evidence to suggest that MGA may constitute a non-obligate precursor of triple-negative breast cancer (TNBC). We sought to define the genomic landscape of pure MGA and of MGA, atypical MGA (AMGA) and associated TNBCs, and to determine whether synchronous MGA, AMGA, and TNBCs would be clonally related. Two pure MGAs and eight cases of MGA and/or AMGA associated with in situ or invasive TNBC were collected, microdissected, and subjected to massively parallel sequencing targeting all coding regions of 236 genes recurrently mutated in breast cancer or related to DNA repair. Pure MGAs lacked clonal non-synonymous somatic mutations and displayed limited copy number alterations (CNAs); conversely, all MGAs (n = 7) and AMGAs (n = 3) associated with TNBC harboured at least one somatic non-synonymous mutation (range 3-14 and 1-10, respectively). In all cases where TNBCs were analyzed, identical TP53 mutations and similar patterns of gene CNAs were found in the MGA and/or AMGA and in the associated TNBC. In the MGA/AMGA associated with TNBC lacking TP53 mutations, somatic mutations affecting PI3K pathway-related genes (eg PTEN, PIK3CA, and INPP4B) and tyrosine kinase receptor signalling-related genes (eg ERBB3 and FGFR2) were identified. At diagnosis, MGAs associated with TNBC were found to display subclonal populations, and clonal shifts in the progression from MGA to AMGA and/or to TNBC were observed. Our results demonstrate the heterogeneity of MGAs, and that MGAs associated with TNBC, but not necessarily pure MGAs, are genetically advanced, clonal, and neoplastic lesions harbouring recurrent mutations in TP53 and/or other cancer genes, supporting the notion that a subset of MGAs and AMGAs may constitute non-obligate precursors of TNBCs.

Synchronous early-stage endometrioid endometrial carcinomas (EECs) and endometrioid ovarian carcinomas (EOCs) are associated with a favorable prognosis and have been suggested to represent independent primary tumors rather than metastatic disease. We subjected sporadic synchronous EECs/EOCs from five patients to whole-exome massively parallel sequencing, which revealed that the EEC and EOC of each case displayed strikingly similar repertoires of somatic mutations and gene copy number alterations. Despite the presence of mutations restricted to the EEC or EOC in each case, we observed that the mutational processes that shaped their respective genomes were consistent. High-depth targeted massively parallel sequencing of sporadic synchronous EECs/EOCs from 17 additional patients confirmed that these lesions are clonally related. In an additional Lynch Syndrome case, however, the EEC and EOC were found to constitute independent cancers lacking somatic mutations in common. Taken together, sporadic synchronous EECs/EOCs are clonally related and likely constitute dissemination from one site to the other.

Aims The aims of this study were to perform a whole-exome sequencing analysis of a breast cylindroma and to investigate the role of molecular analyses in the differentiation between breast cylindroma, a benign tumour that displays MYB expression, and CYLD gene mutations, and its main differential diagnosis, the breast solid-basaloid adenoid cystic carcinoma, a malignant tumour that is characterized by the presence of the MYB–NFIB fusion gene and MYB overexpression. Methods and results A 66-year-old female underwent quadrantectomy after an irregular dense shadow was discovered in the right breast at the screening mammogram. Histologically, the tumour displayed features suggestive of a solid-basaloid variant of adenoid cystic carcinoma with a differential diagnosis of cylindroma. Fluorescence in situ hybridization, reverse transcription–polymerase chain reaction, immunohistochemistry and whole-exome sequencing revealed absence of the MYB–NFIB fusion gene, low levels of MYB protein expression and a clonal somatic CYLD splice site mutation associated with loss of heterozygosity of the wild-type allele. Conclusions The results of the histological, immunohistochemical and molecular analyses were consistent with a diagnosis of breast cylindroma, providing a proof-of-principle that the integration of histopathological and molecular approaches can help to differentiate between a low-malignant potential and a benign breast tumour of triple-negative phenotype.

Triple negative breast cancer represents a heterogeneous group of breast carcinomas, both at the histologic and genetic level. Although recent molecular studies have comprehensively characterized the genetic landscape of these tumors, few have integrated a detailed histologic examination into the analysis. In this study, we defined the genetic alterations in 39 triple negative breast cancers using a high-depth targeted massively parallel sequencing assay and correlated the findings with a detailed morphologic analysis. We obtained representative frozen tissue of primary triple negative breast cancers from patients treated at our institution between 2002 and 2010. We characterized tumors according to their histologic subtype and morphologic features. DNA was extracted from paired frozen primary tumor and normal tissue samples and was subjected to a targeted massively parallel sequencing platform comprising 229 cancer-associated genes common across all experiments. The average number of non-synonymous mutations was 3 (range 0–10) per case. The most frequent somatic alterations were mutations in TP53 (74%) and PIK3CA (10%) and MYC amplifications (26%). Triple negative breast cancers with apocrine differentiation less frequently harbored TP53 mutations (25%) and MYC gains (0%), and displayed a high mutation frequency in PIK3CA and other PI3K signaling pathway-related genes (75%). Using a targeted massively parallel sequencing platform, we identified the key somatic genetic alterations previously reported in triple negative breast cancers. Furthermore, our findings show that triple negative breast cancers with apocrine differentiation constitute a distinct subset, characterized by a high frequency of PI3K pathway alterations similar to luminal subtypes of breast cancer.

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

SummaryPlatelets carry megakaryocyte-derived mRNAs whose translation efficiency before and during activation is not known, although this can greatly affect platelet functions, both under basal conditions and in response to physiological and pathological stimuli, such as those involved in acute coronary syndromes. Aim of the present study was to determine whether changes in microRNA (miRNA) expression occur in response to activating stimuli and whether this affects activity and composition of platelet transcriptome and proteome. Purified platelet-rich plasmas from healthy volunteers were collected and activated with ADP, collagen, or thrombin receptor activating peptide. Transcriptome analysis by RNA-Seq revealed that platelet transcriptome remained largely unaffected within the first 2 hours of stimulation. In contrast, quantitative proteomics showed that almost half of > 700 proteins quantified were modulated under the same conditions. Global miRNA analysis indicated that reorganisation of platelet proteome occurring during activation reflected changes in mature miRNA expression, which therefore, appears to be the main driver of the observed discrepancy between transcriptome and proteome changes. Platelet functions significantly affected by modulated miRNAs include, among others, the integrin/cytoskeletal, coagulation and inflammatory-immune response pathways. These results demonstrate a significant reprogramming of the platelet miRNome during activation, with consequent significant changes in platelet proteome and provide for the first time substantial evidence that fine-tuning of resident mRNA translation by miRNAs is a key event in platelet pathophysiology.

The transcriptome profiles were compared for buffalo embryos with normal growth and embryos with retarded growth on Day 25 after mating. Embryos with retarded growth on Day 25 after mating have a reduced likelihood of undergoing attachment to the uterine endometrium and establishing a pregnancy. Italian Mediterranean buffaloes were mated by AI and on Day 25 underwent trans-rectal ultrasonography to ascertain embryo development. Embryos with an embryonic width (EW)>2.7 mm were classed as normal embryos and embryos with an EW

Background: Massively parallel sequencing studies have identified large numbers of mutations of unknown biologic significance. There is a pressing need for computational methods to predict and distinguish neutral from potentially pathogenic mutations accurately, to help identify those mutations worth exploring experimentally and clinically. Although various bioinformatic algorithms are available, they are based on different methodologies and assumptions, and their predictions for the same mutations are not always concordant. In this study, we sought to benchmark the performance of 17 prediction algorithms using functionally validated and pathognomonic mutations. Methods: We curated the literature for functionally validated and pathognomonic mutations as our positive dataset (i.e. pathogenic mutations). For the negative dataset (i.e. neutral mutations), we retrieved variants from the dbSNP database, including only those with minor allele frequency >25%. We compiled a total of 7975 mutations (875 pathogenic and 7100 neutral). The performance of each prediction algorithm, namely accuracy, specificity, sensitivity, positive predictive value (PPV) and negative predictive value (NPV), were defined using the positive and negative datasets described above. Confidence intervals were calculated by sub-sampling 2/3 of the functionally pathogenic mutations and equal number of neutral mutations 500 times. To reduce the bias introduced by mutations included in the COSMIC database, we excluded those found in COSMIC v67, resulting in 6048 mutations (212 pathogenic and 5835 neutral), and re-evaluated the performance of each prediction algorithm. Results: Our analysis revealed that the overall accuracy varied considerably, with a median of 87% (range 78%-97%). In terms of accuracy, FATHMM (cancer) statistically outperformed all other prediction algorithms (97%, 95% confidence interval (CI) 96%-98%), followed by MutationTaster 2 (94%, 95% CI 93%-95%). Sensitivity and specificity also varied (median 85%, range 77%-96% and median 89%, range 71%-100%, respectively). The most sensitive prediction algorithm, FATHMM (cancer) (96%, 95% CI 95%-97%) statistically outperformed all others. The most specific prediction algorithm was CHASM (breast) (100%, 95% CI 94%-100%). While CHASM (breast) had the highest PPV (100%, 95% CI 99%-100%), FATHMM (cancer) had statistically better NPV than all other prediction algorithms (96%, 95% CI 95%-97%). When COSMIC mutations were removed, FATHMM (cancer) remained the most accurate (93%, 95% CI 91%-95%) though the difference was not statistically significant. In this context, CanDrA (breast) was the most sensitive prediction algorithm (95%, 95% CI 93%-97%) and had the highest NPV (93%, 95% CI 90%-96%), while CHASM (breast) was the most specific prediction algorithm (100%, 95% CI 99%-100%) and had the best PPV (99%, 95% CI 97%-100%). Conclusions: Our results demonstrate that functional prediction algorithms varied in performance. Using this dataset of mutations, FATHMM (cancer) outperformed all other prediction algorithms in terms of accuracy, sensitivity and NPV, and remained the most accurate even when mutations catalogued in the COSMIC database were excluded. Citation Format: Maria R De Filippo, Charlotte KY Ng, Jorge S Reis-Filho, Britta Weigelt. Benchmarking mutation function prediction algorithms using validated cancer driver and passenger mutations [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-03-09.

Introduction: Metaplastic breast carcinoma (MBC) is an aggressive histologic type of breast cancer, which preferentially displays a triple-negative phenotype. These tumors are characterized by the presence of malignant cells exhibiting differentiation towards squamous epithelium or mesenchymal elements, including spindle, chondroid, osseous and rhabdoid differentiation. Unlike other rare histologic types of breast cancer such as adenoid cystic and secretory carcinomas, which are underpinned by the MYB-NFIB and ETV6-NTRK3 fusion genes respectively, pathognomonic genetic alterations have not been identified in MBC. It has been suggested, however, that the frequency of PIK3CA somatic mutations would be significantly higher in MBCs than in other forms of triple-negative disease. Here we sought to characterize the mutational landscape of MBCs by means of high-depth whole exome sequencing analysis. Material and Methods: Twenty-one triple-negative MBCs were retrieved from the authors’ institutions. Representative sections from frozen blocks were microdissected to ensure tumor cell content greater than 50%. DNA samples extracted from microdissected tumor and matched peripheral blood leukocytes were subjected to high-depth (250x) whole exome sequencing on an Illumina GAIIx or HiSeq2000. Somatic point mutations were called using MuTect and somatic insertions and deletions (indels) were called using Strelka, Varscan2 and Haplotype Caller. Potentially pathogenic mutations were predicted using computational algorithms including PolyPhen-2, Mutation Taster, Mutation Assessor, CHASM and FATHMM. Significantly mutated genes were identified using MutSigCV. Pathway and network enrichment analysis of mutations was performed with Ingenuity Pathway Analysis and HOTNET. The genomic landscape of MBCs was compared with that of triple-negative breast cancers (TNBCs) analyzed as part of The Cancer Genome Atlas project. Results: A mean of 135 somatic non-synonymous point mutations and indels were identified per MBC. The most frequently mutated gene was TP53, found in 12/21 cases (57%), and the only significantly mutated gene as defined by MutSigCV (q Conclusion: The majority (57%) of MBCs harbored non-synonymous mutations affecting TP53. While the frequencies of mutations affecting recurrently mutated genes in MBCs are similar to those found in other forms of TNBCs, MBCs significantly more frequently harbor mutations affecting PI3K pathway-related genes than TNBCs of no special type. Citation Format: Charlotte KY Ng, Britta Weigelt, Salvatore Piscuoglio, Y Hannah Wen, Maria R De Filippo, Luciano G Martelotto, Rachael Natrajan, Raymond Lim, Edi Brogi, Larry Norton, Anne Vincent-Salomon, Jorge S Reis-Filho. Mutational landscape of metaplastic breast carcinomas [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-03-08.

Background Qualitative and quantitative analysis of small non-coding RNAs by next generation sequencing (smallRNA-Seq) represents a novel technology increasingly used to investigate with high sensitivity and specificity RNA population comprising microRNAs and other regulatory small transcripts. Analysis of smallRNA-Seq data to gather biologically relevant information, i.e. detection and differential expression analysis of known and novel non-coding RNAs, target prediction, etc., requires implementation of multiple statistical and bioinformatics tools from different sources, each focusing on a specific step of the analysis pipeline. As a consequence, the analytical workflow is slowed down by the need for continuous interventions by the operator, a critical factor when large numbers of datasets need to be analyzed at once. Results We designed a novel modular pipeline (iMir) for comprehensive analysis of smallRNA-Seq data, comprising specific tools for adapter trimming, quality filtering, differential expression analysis, biological target prediction and other useful options by integrating multiple open source modules and resources in an automated workflow. As statistics is crucial in deep-sequencing data analysis, we devised and integrated in iMir tools based on different statistical approaches to allow the operator to analyze data rigorously. The pipeline created here proved to be efficient and time-saving than currently available methods and, in addition, flexible enough to allow the user to select the preferred combination of analytical steps. We present here the results obtained by applying this pipeline to analyze simultaneously 6 smallRNA-Seq datasets from either exponentially growing or growth-arrested human breast cancer MCF-7 cells, that led to the rapid and accurate identification, quantitation and differential expression analysis of ~450 miRNAs, including several novel miRNAs and isomiRs, as well as identification of the putative mRNA targets of differentially expressed miRNAs. In addition, iMir allowed also the identification of ~70 piRNAs (piwi-interacting RNAs), some of which differentially expressed in proliferating vs growth arrested cells. Conclusion The integrated data analysis pipeline described here is based on a reliable, flexible and fully automated workflow, useful to rapidly and efficiently analyze high-throughput smallRNA-Seq data, such as those produced by the most recent high-performance next generation sequencers. iMir is available at http://www.labmedmolge.unisa.it/inglese/research/imir.

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.

Estrogen receptor alpha (ERα) is a ligand-activated transcription factor that controls key cellular pathways via protein-protein interactions involving multiple components of transcriptional coregulator and signal transduction complexes. Natural and synthetic ERα ligands are classified as agonists (17β-estradiol/E(2)), selective estrogen receptor modulators (SERMs: Tamoxifen/Tam and Raloxifene/Ral), and pure antagonists (ICI 182,780-Fulvestrant/ICI), according to the response they elicit in hormone-responsive cells. Crystallographic analyses reveal ligand-dependent ERα conformations, characterized by specific surface docking sites for functional protein-protein interactions, whose identification is needed to understand antiestrogen effects on estrogen target tissues, in particular breast cancer (BC). Tandem affinity purification (TAP) coupled to mass spectrometry was applied here to map nuclear ERα interactomes dependent upon different classes of ligands in hormone-responsive BC cells. Comparative analyses of agonist (E(2))- vs antagonist (Tam, Ral or ICI)-bound ERα interacting proteins reveal significant differences among ER ligands that relate with their biological activity, identifying novel functional partners of antiestrogen-ERα complexes in human BC cell nuclei. In particular, the E(2)-dependent nuclear ERα interactome is different and more complex than those elicited by Tam, Ral, or ICI, which, in turn, are significantly divergent from each other, a result that provides clues to explain the pharmacological specificities of these compounds.

Oestrogen receptor alpha (ERα) is a ligand-dependent transcription factor that mediates oestrogen effects in hormone-responsive cells. Following oestrogenic activation, ERα directly regulates the transcription of target genes via DNA binding. MicroRNAs (miRNAs) represent a class of small noncoding RNAs that function as negative regulators of protein-coding gene expression. They are found aberrantly expressed or mutated in cancer, suggesting their crucial role as either oncogenes or tumour suppressor genes. Here, we analysed changes in miRNA expression in response to oestrogen in hormone-responsive breast cancer MCF-7 and ZR-75.1 cells by microarray-mediated expression profiling. This led to the identification of 172 miRNAs up- or down-regulated by ERα in response to 17β-oestradiol, of which 52 are similarly regulated by the hormone in the two cell models investigated. To identify mechanisms by which ERα exerts its effects on oestrogen-responsive miRNA genes, the oestrogen-dependent miRNA expression profiles were integrated with global in vivo ERα binding site mapping in the genome by ChIP-Seq. In addition, data from miRNA and messenger RNA (mRNA) expression profiles obtained under identical experimental conditions were compared to identify relevant miRNA target transcripts. Results show that miRNAs modulated by ERα represent a novel genomic pathway to impact oestrogen-dependent processes that affect hormone-responsive breast cancer cell behaviour. MiRNome analysis in tumour tissues from breast cancer patients confirmed a strong association between expression of these small RNAs and clinical outcome of the disease, although this appears to involve only marginally the oestrogen-regulated miRNAs identified in this study.

Background Estrogen receptors alpha (ERα) and beta (ERβ) are transcription factors (TFs) that mediate estrogen signaling and define the hormone-responsive phenotype of breast cancer (BC). The two receptors can be found co-expressed and play specific, often opposite, roles, with ERβ being able to modulate the effects of ERα on gene transcription and cell proliferation. ERβ is frequently lost in BC, where its presence generally correlates with a better prognosis of the disease. The identification of the genomic targets of ERβ in hormone-responsive BC cells is thus a critical step to elucidate the roles of this receptor in estrogen signaling and tumor cell biology. Results Expression of full-length ERβ in hormone-responsive, ERα-positive MCF-7 cells resulted in a marked reduction in cell proliferation in response to estrogen and marked effects on the cell transcriptome. By ChIP-Seq we identified 9702 ERβ and 6024 ERα binding sites in estrogen-stimulated cells, comprising sites occupied by either ERβ, ERα or both ER subtypes. A search for TF binding matrices revealed that the majority of the binding sites identified comprise one or more Estrogen Response Element and the remaining show binding matrixes for other TFs known to mediate ER interaction with chromatin by tethering, including AP2, E2F and SP1. Of 921 genes differentially regulated by estrogen in ERβ+ vs ERβ- cells, 424 showed one or more ERβ site within 10 kb. These putative primary ERβ target genes control cell proliferation, death, differentiation, motility and adhesion, signal transduction and transcription, key cellular processes that might explain the biological and clinical phenotype of tumors expressing this ER subtype. ERβ binding in close proximity of several miRNA genes and in the mitochondrial genome, suggests the possible involvement of this receptor in small non-coding RNA biogenesis and mitochondrial genome functions. Conclusions Results indicate that the vast majority of the genomic targets of ERβ can bind also ERα, suggesting that the overall action of ERβ on the genome of hormone-responsive BC cells depends mainly on the relative concentration of both ERs in the cell.

Background: Endometrioid endometrial carcinomas (EECs) frequently harbor mutations in the PI3K pathway. In contrast with other cancer types (e.g. breast cancer) where PIK3CA mutations are generally mutually exclusive with PTEN mutations, in EECs mutations affecting these genes often co-occur. Here we sought to determine whether the type and pattern of mutations targeting different components of the PI3K pathway are distinct between microsatellite stable (MSS) and high-level microsatellite instable (MSI-H) EECs, and to define the mutational signatures in MSI-H and MSS EECs. Methods: Whole exome massively parallel sequencing-based mutation data from EECs of The Cancer Genome Atlas (TCGA) project were used to define the number, type and pattern of mutations affecting PI3K pathway-related genes (i.e., AKT1, INPP4B, MTOR, PIK3CA, PIK3R1 and PTEN). Based on seven MSI markers assessed by TCGA, EECs were classified as MSI-H (n = 70) and MSS (n = 109). POLE ultramutated cases were excluded. Mutational signatures were defined using EMu, a method based upon the expectation-maximization algorithm. Results: Although the mutation rates of MSS and MSI-H EECs were significantly different, the prevalence of mutations affecting PI3K pathway genes was similar between these two groups (all p>0.05), with the exception of PTEN mutations, which were more prevalent in MSI-H (87%) than in MSS EECs (72%; p = 0.017). The PIK3CA hotspot mutations E542K, E545K, and H1047R were found to be significantly more prevalent in PIK3CA-mutant MSS EECs (36%) than in PIK3CA-mutant MSI-H EECs (13.5%; p = 0.019). In both MSI-H and MSS EECs a mutational signature related to age was identified, characterized by C>T transitions at NpCpG trinucleotides; in MSS tumors a C>T and C>G at TpCpN trinucleotides mutational signature, attributed to the APOBEC family of cytidine deaminases, was identified, whereas in MSI-H tumors, a DNA-MMR deficiency-like signature was found. Conclusion: Although the prevalence of mutations targeting different components of the PI3K pathway is similar between MSS and MSI-H EECs, PIK3CA hotspot mutations, which result in constitutive kinase activation, are significantly more prevalent in MSS than in MSI-H EECs. We have observed that the mutational processes operating in MSI-H and MSS EECs are distinct, and that the landscape of mutations affecting PI3K pathway-related genes might be shaped by multiple mutational processes in these cancers. Our findings warrant further investigation of the role of different types of PIK3CA mutations in and their predictive impact on distinct subtypes of EECs. Citation Format: Caterina Marchio, Maria R. De Filippo, Charlotte KY Ng, Robert A. Soslow, Jorge S. Reis-Filho, Britta Weigelt. Microsatellite instability status in endometrioid endometrial carcinomas is associated with distinct types and patterns of PI3K pathway mutations. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4817. doi:10.1158/1538-7445.AM2015-4817

Background Massively parallel sequencing studies have led to the identification of a large number of mutations present in a minority of cancers of a given site. Hence, methods to identify the likely pathogenic mutations that are worth exploring experimentally and clinically are required. We sought to compare the performance of 15 mutation effect prediction algorithms and their agreement. As a hypothesis-generating aim, we sought to define whether combinations of prediction algorithms would improve the functional effect predictions of specific mutations. Results Literature and database mining of single nucleotide variants (SNVs) affecting 15 cancer genes was performed to identify mutations supported by functional evidence or hereditary disease association to be classified either as non-neutral (n = 849) or neutral (n = 140) with respect to their impact on protein function. These SNVs were employed to test the performance of 15 mutation effect prediction algorithms. The accuracy of the prediction algorithms varies considerably. Although all algorithms perform consistently well in terms of positive predictive value, their negative predictive value varies substantially. Cancer-specific mutation effect predictors display no-to-almost perfect agreement in their predictions of these SNVs, whereas the non-cancer-specific predictors showed no-to-moderate agreement. Combinations of predictors modestly improve accuracy and significantly improve negative predictive values. Conclusions The information provided by mutation effect predictors is not equivalent. No algorithm is able to predict sufficiently accurately SNVs that should be taken forward for experimental or clinical testing. Combining algorithms aggregates orthogonal information and may result in improvements in the negative predictive value of mutation effect predictions. Electronic supplementary material The online version of this article (doi:10.1186/s13059-014-0484-1) contains supplementary material, which is available to authorized users.