Pan-cancer evaluation of tumor-infiltrating lymphocytes and programmed cell death protein ligand-1 in metastatic biopsies and matched primary tumors.

Tumor immunological characterization includes evaluation of tumor-infiltrating lymphocytes (TILs) and programmed cell death protein ligand-1 (PD-L1) expression. This study investigated TIL distribution, its prognostic value, and PD-L1 expression in metastatic and matched primary tumors (PTs). Specimens from 550 pan-cancer patients of the SHIVA01 trial (NCT01771458) with available metastatic biopsy and 111 matched PTs were evaluated for TILs and PD-L1. Combined positive score (CPS), tumor proportion score (TPS), and immune cell (IC) score were determined. TILs and PD-L1 were assessed according to PT organ of origin, histological subtype, and metastatic biopsy site. We found that TIL distribution in metastases did not vary according to PT organ of origin, histological subtype, or metastatic biopsy site, with a median of 10% (range: 0-70). TILs were decreased in metastases compared to PT (20% [5-60] versus 10% [0-40], p < 0.0001). CPS varied according to histological subtype (p = 0.02) and biopsy site (p < 0.02). TPS varied according to PT organ of origin (p = 0.003), histological subtype (p = 0.0004), and metastatic biopsy site (p = 0.00004). TPS was higher in metastases than in PT (p < 0.0001). TILs in metastases did not correlate with overall survival. In conclusion, metastases harbored fewer TILs than matched PT, regardless of PT organ of origin, histological subtype, and metastatic biopsy site. PD-L1 expression increased with disease progression. © 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

E-Cadherin Mutational Landscape and Outcomes in Breast Invasive Lobular Carcinoma.

Invasive lobular carcinomas (ILC) are characterized by the loss of E-cadherin expression and CDH1 gene inactivation. Diagnostic reproducibility for this tumor type is currently suboptimal and could be improved by a better understanding of its histomolecular and clinical heterogeneity. We have analyzed the relationship between the presence, type, or position of CDH1 mutations, E-cadherin expression, and clinicopathological features (including outcome) in a retrospective series of 251 primary ILC with a long follow-up (median: 9.5 years). The mutational status of E-cadherin gene (CDH1) was determined by RNA sequencing from frozen tumor samples. E-cadherin immunohistochemistry (IHC) was performed with antibodies directed against the intracellular domain (clone 4A2C7) and the extracellular domain (clone NCH38). IHC expression of p120 and ß-catenin was also assessed in E-cadherin diffusely positive cases. Three major patterns of E-cadherin membrane expression were identified by IHC, with good agreement between the 2 clones (overall concordance: 83.8%, Kappa 0.67): null/focal expression (≤10%) (72.8% of cases for 4A2C7 and 83.8% for NCH38), heterogeneous expression (11%-89%) (19.2% of cases for 4A2C7 and 6.9% for NCH38), and diffuse expression (≥90%) (8% of cases for 4A2C7 and 9.3% for NCH38). E-cadherin membranous expression, when present, was abnormal (incomplete labeling and/or reduced intensity). ILC with diffuse E-cadherin expression showed abnormal ß-catenin or p120-catenin staining in 21% of cases. Interestingly, these cases with diffusely expressed E-cadherin had a CDH1 mutation rate as high as the E-cadherin null/focal cases (∼70%) but were enriched in nontruncating mutations. Regarding CDH1 mutation location, intracytoplasmic domain mutations correlated with a divergent E-cadherin IHC phenotype between the 2 antibodies (4A2C7 ≤ 10%/NCH38 ≥ 10%). Clinico-pathological correlation analyses found that stromal amount (inversely correlated with tumor cellularity) and tumor-infiltrating lymphocytes were less abundant in ILC with E-cadherin null/focal cases. In addition, CDH1 truncating mutations were associated with radiohistologic size discordance and were identified in multivariate survival analysis as an independent poor prognosis factor in terms of metastasis risk and breast cancer-related mortality. Overall, our study highlights the importance of the precise mutational status of CDH1 in the clinical, radiological, histologic, and phenotypic expression of lobular carcinomas. These findings should be taken into account in future attempts to improve diagnostic criteria or methods for ILC, as well as for clinicobiological studies dedicated to this tumor type.

Molecular and Clinical Portrait of HER2-low Invasive Lobular Carcinomas.

Invasive lobular carcinomas (ILCs) have a low frequency of ERBB2 amplification, therefore restricting the use of conventional anti-HER2 therapies for this histologic special type. Conversely, ILCs with low HER2 overexpression may represent a broader target for the use of emerging antibody drug conjugate therapies targeting HER2, since these treatments have proven effective in HER2-low breast cancers. Very scarce data about HER2-low ILCs have been so far published, although these tumors could have different prevalence and histomolecular specificities compared with invasive breast carcinoma of no special type (IBC-NST). Our aims in that context were to decipher the clinicopathological and molecular features of a large series of HER2-low ILCs. Comparative evaluation of HER2-low prevalence was done based on a retrospective series of 7970 patients from Institut Curie, with either primary invasive lobular (N = 1103) or no special type (N = 6867) invasive carcinoma. Clinicopathological and molecular analyses of HER2-zero, HER2-low, and HER2-positive ILCs were performed on a subgroup of 251 patients who underwent surgery for a primary ILC between 2005 and 2008. The mutational profile of these 251 cases was determined from RNAseq data. Compared with HER2-negative IBC-NSTs, the HER2-negative ILCs were found to display a higher frequency of HER2-zero cases (59.4% vs 53.7%) and a lower frequency of HER2-low (40.6% vs 46.3%) (P < .001). Clinicopathological features associated with HER2-low status (vs HER2-zero) in ILC were older age, postmenopausal status, nonclassic ILC histological types, higher grade, proliferation, and estrogen receptor expression levels. Survival curve analysis showed a significantly lower risk of local recurrence for HER2-low (vs HER2-zero) ILCs, but no association was found between HER2 status and either breast cancer-specific survival or distant metastasis-free interval. ERBB3 was the unique mutated gene exclusively associated with HER2-low ILCs yet being mutated at a low frequency (7.1%) (false discovery rate < 0.05). In conclusion, HER2-low ILCs exhibit their own particularities, both on clinical-pathological and molecular levels. Our findings call for larger multicenter validation studies.

MDM4 amplification in atypical lipomatous tumors/well-differentiated liposarcoma: Private event or alternative oncogenic mechanism?

Adipocytic tumors are the most common mesenchymal tumors in soft tissues. Among them, a diagnostic challenge relies in the distinction between lipoma and atypical lipomatous tumor (ALT)/well differentiated liposarcoma (WDLPS), as both entities are often undistinguishable not only from a radiological point of view, but also at the microscopic level and particularly when dealing with small tumor specimen. Thus, detection of recurrent MDM2 amplifications may be the only criteria to discriminate malignant tumors from lipomas. In this study, we report the case of a patient diagnosed with a well differentiated, adipocytic tumor located in the inferior limb and lacking MDM2 amplification, whose diagnosis was reclassified for ALT/WDLPS after identification of an alternative MDM4 amplification by comparative genomic hybridization profiling, whole exome sequencing and fluorescence in situ hybridization (FISH). Screening of a cohort of 37 large, deep-seated, well-differentiated adipocytic tumors previously classified as lipomas using RT-qPCR and FISH failed to detect other cases of MDM4-amplified ALT/WDLPS. This report shows that MDM4 amplification is an exceptional molecular event alternative to MDM2 amplification in ALT/WDLPS. This alteration should be considered and looked for in suspicious adipocytic tumors to optimize their surgical management.

Breast carcinomas with osteoclast-like giant cells: a comprehensive clinico-pathological and molecular portrait and evidence of RANK-L expression.

Breast carcinomas (BC) with osteoclast-like giant cells (OGC) are rare. Despite their distinct stromal features, their molecular characteristics remain unknown. Here, we report comprehensive clinico-pathological and molecular findings for 27 patients diagnosed with BC-OGC at Institut Curie between 2000 and 2021. Seventeen (63%) cases were invasive carcinomas of no special type (IC NST) with OGC (OGC-IC NST), four (15%) were mixed or multifocal cases with and without OGC (OGC-Mixed), and six (22%) were metaplastic carcinomas with OGC (OGC-MC). All OGC-IC NST and OGC-Mixed cases were ER+ HER2- tumors (most being luminal A based on transcriptomic subtyping, when available), while all OGC-MC were triple-negative. The median age at diagnosis was 46, 45 and 62 years for OGC-IC NST, OGC-Mixed and OGC-MC, respectively. Three patients developed distant metastases (one OGC-IC NST, two OGC-Mixed), one of whom died of metastatic disease (OGC-Mixed), and one other patient died of locally advanced disease (OGC-MC). Histopathological evaluation comparing 13 OGC-IC NST and 19 control IC NST without OGC confirmed that OGC-IC NST showed significantly higher density of vessels (by CD34 immunohistochemistry (IHC)), iron deposits (Perls stain), and CD68 and CD163-positive cell infiltrates. Genomic findings for nine OGC-IC NST and four OGC-MC were consistent with the underlying histologic subtype, including activating alterations of the PI3K/AKT/mTOR pathway in 7/13 cases. Using RNA-seq data, differential gene expression analysis between OGC-IC NST (n = 7) and control IC NST without OGC (n = 7) revealed significant overexpression of TNFSF11 (RANK-L), TNFRSF11A (RANK), CSF1 (M-CSF), CSF1R, and genes encoding osteoclastic enzymes (MMP9, ACP5, CTSK, CTSB) in OGC-IC NST, while OPG (osteoprotegerin) was underexpressed. We also confirmed for the first time RANK-L expression in BC with OGC by IHC (seen in 15 out of 16 cases, and only in 2 of 16 controls without OGC). These findings could offer a rationale for further investigating RANK-L as a therapeutic target in BC with OGC.

Assessment of the Molecular Heterogeneity of E-Cadherin Expression in Invasive Lobular Breast Cancer.

Mutations and loss of E-cadherin protein expression define the vast majority of invasive lobular carcinomas. In a subset of these cases, the heterogeneous expression of E-cadherin is observed either as wild-type (strong membranous) expression or aberrant expression (cytoplasmic expression). However, it is unclear as to whether the two components would be driven by distinct genetic or epigenetic alterations. Here, we used whole genome DNA sequencing and methylation array profiling of two separately dissected components of nine invasive lobular carcinomas with heterogeneous E-cadherin expression. E-cadherin negative and aberrant/positive components of E-cadherin heterogeneous tumours showed a similar mutational, copy number and promoter methylation repertoire, suggesting they arise from a common ancestor, as opposed to the collision of two independent tumours. We found that the majority of E-cadherin heterogeneous tumours harboured CDH1 mutations in both the E-cadherin negative and aberrant/positive components together with somatic mutations in additional driver genes known to be enriched in both pure invasive carcinomas of no special type and invasive lobular breast cancers, whereas these were less commonly observed in CDH1 wild-type tumours. CDH1 mutant tumours also exhibited a higher mutation burden as well as increased presence of APOBEC-dependent mutational signatures 2 and 13 compared to CDH1 wild-type tumours. Together, our results suggest that regardless of E-cadherin protein expression, tumours showing heterogeneous expression of E-cadherin should be considered as part of the spectrum of invasive lobular breast cancers.

Metastasis-suppressor NME1 controls the invasive switch of breast cancer by regulating MT1-MMP surface clearance.

Membrane Type 1 Matrix Metalloprotease (MT1-MMP) contributes to the invasive progression of breast cancers by degrading extracellular matrix tissues. Nucleoside diphosphate kinase, NME1/NM23-H1, has been identified as a metastasis suppressor; however, its contribution to local invasion in breast cancer is not known. Here, we report that NME1 is up-regulated in ductal carcinoma in situ (DCIS) as compared to normal breast epithelial tissues. NME1 levels drop in microinvasive and invasive components of breast tumor cells relative to synchronous DCIS foci. We find a strong anti-correlation between NME1 and plasma membrane MT1-MMP levels in the invasive components of breast tumors, particularly in aggressive histological grade III and triple-negative breast cancers. Knockout of NME1 accelerates the invasive transition of breast tumors in the intraductal xenograft model. At the mechanistic level, we find that MT1-MMP, NME1 and dynamin-2, a GTPase known to require GTP production by NME1 for its membrane fission activity in the endocytic pathway, interact in clathrin-coated vesicles at the plasma membrane. Loss of NME1 function increases MT1-MMP surface levels by inhibiting endocytic clearance. As a consequence, the ECM degradation and invasive potentials of breast cancer cells are enhanced. This study identifies the down-modulation of NME1 as a potent driver of the in situ-to invasive transition during breast cancer progression.

EHD2 is a Predictive Biomarker of Chemotherapy Efficacy in Triple Negative Breast Carcinoma.

EHD2 is a mechanotransducing ATPase localized in caveolae invaginations at the plasma membrane. EHD2 has recently been associated with several human cancers, however the significance of EHD2 transcript levels in cancer prognosis remains debated. Breast cancer is the most commonly occurring cancer in women and prognosis is variable depending on the subtypes. Triple negative breast cancer (TNBC) often has a poor therapeutic response. The aim of this study was to assess the prognostic significance of EHD2 transcripts and protein expression levels in breast carcinomas. We found that low EHD2 levels were associated with enhanced proliferation, migration and invasion of TNBC cells. EHD2 expression was significantly reduced in TNBC tissues and the loss of EHD2 led to higher expression of the pro-tumoral cytokine IL-8. In apparent contradiction with in vitro data, multivariate analysis of two independent cohorts of breast cancer patients revealed that low EHD2 was in fact associated with good prognosis in the highly proliferative TNBC subtype. Accordingly, TNBC low EHD2 expressers were found to benefit the most from chemotherapy when compared to all subtypes of breast cancers. Our study validates EHD2 expression level as an independent prognostic factor of metastasis-free survival and as a new predictive marker of chemotherapy efficacy in TNBC patients.

BRCAness, SLFN11, and RB1 loss predict response to topoisomerase I inhibitors in triple-negative breast cancers.

Topoisomerase I (TOP1) inhibitors trap TOP1 cleavage complexes resulting in DNA double-strand breaks (DSBs) during replication, which are repaired by homologous recombination (HR). Triple-negative breast cancer (TNBC) could be eligible for TOP1 inhibitors given the considerable proportion of tumors with a defect in HR-mediated repair (BRCAness). The TOP1 inhibitor irinotecan was tested in 40 patient-derived xenografts (PDXs) of TNBC. BRCAness was determined with a single-nucleotide polymorphism (SNP) assay, and expression of Schlafen family member 11 (SLFN11) and retinoblastoma transcriptional corepressor 1 (RB1) was evaluated by real-time polymerase chain reaction (RT-PCR) and immunohistochemistry analyses. In addition, the combination of irinotecan and the ataxia telangiectasia and Rad3-related protein (ATR) inhibitor VE-822 was tested in SLFN11-negative PDXs, and two clinical non-camptothecin TOP1 inhibitors (LMP400 and LMP776) were tested. Thirty-eight percent of the TNBC models responded to irinotecan. BRCAness combined with high SLFN11 expression and RB1 loss identified highly sensitive tumors, consistent with the notion that deficiencies in cell cycle checkpoints and DNA repair result in high sensitivity to TOP1 inhibitors. Treatment by the ATR inhibitor VE-822 increased sensitivity to irinotecan in SLFN11-negative PDXs and abolished irinotecan-induced phosphorylation of checkpoint kinase 1 (CHK1). LMP400 (indotecan) and LMP776 (indimitecan) showed high antitumor activity in BRCA1-mutated or BRCAness-positive PDXs. Last, low SLFN11 expression was associated with poor survival in 250 patients with TNBC treated with anthracycline-based chemotherapy. In conclusion, a substantial proportion of TNBC respond to irinotecan. BRCAness, high SLFN11 expression, and RB1 loss are highly predictive of response to irinotecan and the clinical indenoisoquinoline TOP1 inhibitors.

Cancer-associated fibroblast heterogeneity in axillary lymph nodes drives metastases in breast cancer through complementary mechanisms.

Although fibroblast heterogeneity is recognized in primary tumors, both its characterization in and its impact on metastases remain unknown. Here, combining flow cytometry, immunohistochemistry and RNA-sequencing on breast cancer samples, we identify four Cancer-Associated Fibroblast (CAF) subpopulations in metastatic lymph nodes (LN). Two myofibroblastic subsets, CAF-S1 and CAF-S4, accumulate in LN and correlate with cancer cell invasion. By developing functional assays on primary cultures, we demonstrate that these subsets promote metastasis through distinct functions. While CAF-S1 stimulate cancer cell migration and initiate an epithelial-to-mesenchymal transition through CXCL12 and TGFß pathways, highly contractile CAF-S4 induce cancer cell invasion in 3-dimensions via NOTCH signaling. Patients with high levels of CAFs, particularly CAF-S4, in LN at diagnosis are prone to develop late distant metastases. Our findings suggest that CAF subset accumulation in LN is a prognostic marker, suggesting that CAF subsets could be examined in axillary LN at diagnosis.

Pan-TRK Immunohistochemistry: A Useful Diagnostic Adjunct For Secretory Carcinoma of the Breast.

Secretory carcinoma is a special-type breast carcinoma underpinned by a recurrent t(12;15)(p13;q25) translocation resulting in ETV6-NTRK3 gene fusion. Immunohistochemistry (IHC) using a pan-TRK antibody has been recently shown to help identify NTRK rearrangements in other tumor types. The purpose of this study was to assess the diagnostic utility of pan-TRK IHC in secretory carcinoma of the breast. Pan-TRK IHC was performed using a rabbit monoclonal antibody on whole sections of 24 breast secretory carcinomas and tissue microarray sections of other breast carcinoma types (n=203) and histologic mimics (n=15). Cases were assessed for staining intensity and localization. The 24 patients with secretory carcinoma had a median age of 44 years and a median tumor size of 1.0 cm. ETV6 fluorescence in situ hybridization was positive in all cases tested (n=20). Twenty-three cases (95.8%) showed staining with pan-TRK, which was exclusively nuclear in 19, primarily nuclear with weak cytoplasmic staining in 3, and primarily cytoplasmic with focal nuclear staining in 1. The nuclear staining was diffuse in 17 and at least focally strong in 17. The only pan-TRK negative case was a core biopsy with limited tumor. Among the 203 nonsecretory carcinomas, 21 (10.3%) showed focal, weak nuclear staining in <5% of tumor cells and 1 (0.5%) showed focal membranous staining. All histologic mimics were negative. In conclusion, diffuse and/or at least focally strong nuclear pan-TRK staining is a sensitive and specific marker for secretory carcinoma of the breast.

The Genomic Landscape of Mucinous Breast Cancer.

Mucinous carcinoma of the breast (MCB) is a rare histologic form of estrogen receptor (ER)-positive/HER2-negative breast cancer (BC) characterized by tumor cells floating in lakes of mucin. We assessed the genomic landscape of 32 MCBs by whole-exome sequencing and/or RNA-sequencing. GATA3 (23.8%), KMT2C (19.0%), and MAP3K1 (14.3%) were the most frequently mutated genes in pure MCBs. In addition, two recurrent but not pathognomonic fusion genes, OAZ1-CSNK1G2 and RFC4-LPP, were detected in 3/31 (9.7%) and 2/31 (6.5%) samples, respectively. Compared with ER-positive/HER2-negative common forms of BC, MCBs displayed lower PIK3CA and TP53 mutation rates and fewer concurrent 1q gains and 16q losses. Clonal decomposition analysis of the mucinous and ductal components independently microdissected from five mixed MCBs revealed that they are clonally related and evolve following clonal selection or parallel evolution. Our findings indicate that MCB represents a genetically distinct ER-positive/HER2-negative form of BC.

Combinatorial expression of microtubule-associated EB1 and ATIP3 biomarkers improves breast cancer prognosis.

PURPOSE: The identification of molecular biomarkers for classification of breast cancer is needed to better stratify the patients and guide therapeutic decisions. The aim of this study was to investigate the value of MAPRE1 gene encoding microtubule-end binding proteins EB1 as a biomarker in breast cancer and evaluate whether combinatorial expression of MAPRE1 and MTUS1 gene encoding EB1-negative regulator ATIP3 may improve breast cancer diagnosis and prognosis. METHODS: Probeset intensities for MAPRE1 and MTUS1 genes were retrieved from Exonhit splice array analyses of 45 benign and 120 malignant breast tumors for diagnostic purposes. Transcriptomic analyses (U133 Affymetrix array) of one exploratory cohort of 150 invasive breast cancer patients and two independent series of 130 and 155 samples were compared with clinical data of the patients for prognostic studies. A tissue microarray from an independent cohort of 212 invasive breast tumors was immunostained with anti-EB1 and anti-ATIP3 antibodies. RESULTS: We show that MAPRE1 gene is a diagnostic and prognostic biomarker in breast cancer. High MAPRE1 levels correlate with tumor malignancy, high histological grade and poor clinical outcome. Combination of high-MAPRE1 and low-MTUS1 levels in tumors is significantly associated with tumor aggressiveness and reduced patient survival. IHC studies of combined EB1/ATIP3 protein expression confirmed these results. CONCLUSIONS: These studies emphasize the importance of studying combinatorial expression of EB1 and ATIP3 genes and proteins rather than each biomarker alone. A population of highly aggressive breast tumors expressing high-EB1/low-ATIP3 may be considered for the development of new molecular therapies.

Distinct expression profiles and functions of Kindlins in breast cancer.

BACKGROUND: Kindlin-1, - 2, and - 3 are the three members of the Kindlin family. They are best known as regulators of integrin functions, contributing to fundamental biological processes such as cell survival, adhesion and migration. Their deregulation leads to diverse pathologies including a broad range of cancers in which both, tumor-promoting and tumor-inhibiting functions have been described. METHODS: To better characterize Kindlins implication in breast cancer, in vitro experiments were performed in a series of cancer cell lines. We first assessed their expression profiles and subcellular distributions. Then, their involvement in breast cancer cell morphology, migration and invasion was verified by examining phenotypic changes induced by the depletion of either isoforms using RNA interference. An expression study was performed in a series of breast cancer patient derived xenografts (n = 58) to define the epithelial and stromal contribution of each Kindlin. Finally, we analyzed the expression levels of the three Kindlins in a large series of human breast tumors, at the RNA (n = 438) and protein (n = 129) levels and we evaluated their correlation with the clinical outcome. RESULTS: We determined that Kindlin-1 and Kindlin-2, but not Kindlin-3, were expressed in breast tumor cells. We uncovered the compensatory roles of Kindlin-1 and -2 in focal adhesion dynamics and cell motility. Remarkably, Kindlin-2 had a predominant effect on cell spreading and Kindlin-1 on cell invasion. In line with these experimental observations, Kindlin-1 overexpression was associated with a worse patients' outcome. Notably, Kindlin-3, expressed by tumor infiltrating leukocytes, also correlated with a poor prognosis of breast cancer patients. CONCLUSION: This study demonstrates that each one of the Kindlin family members has a different expression profile emphasizing their redundant and complementary roles in breast tumor cells. We highlight the specific link between Kindlin-1 and breast cancer progression. In addition, Kindlin-3 overexpression in the tumor microenvironment is associated with more aggressive breast tumors. These results suggest that Kindlins play distinctive roles in breast cancer. Kindlins may be useful in identifying breast cancer patients with a worst prognosis and may offer new avenues for therapeutic intervention against cancer progression.

VOPP1 promotes breast tumorigenesis by interacting with the tumor suppressor WWOX.

BACKGROUND: The WW domain-containing oxidoreductase (WWOX) gene, frequently altered in breast cancer, encodes a tumor suppressor whose function is mediated through its interactions with cancer-related proteins, such as the pro-apoptotic protein p73α. RESULTS: To better understand the involvement of WWOX in breast tumorigenesis, we performed a yeast two-hybrid screen and co-immunoprecipitation assays to identify novel partners of this protein. We characterized the vesicular overexpressed in cancer pro-survival protein 1 (VOPP1) as a new regulator of WWOX. In breast cancer cells, VOPP1 sequestrates WWOX in lysosomes, impairs its ability to associate with p73α, and inhibits WWOX-dependent apoptosis. Overexpressed VOPP1 potentiates cellular transformation and enhances the growth of transplanted tumors in vivo. VOPP1 is overexpressed in breast tumors, especially in tumors that retain WWOX. Moreover, increased expression of VOPP1 is associated with reduced survival of patients with WWOX-positive, but not with WWOX-negative, tumors. CONCLUSIONS: These findings emphasize the importance of the sequestration of WWOX by VOPP1 in addition to WWOX loss in breast tumors and define VOPP1 as a novel oncogene promoting breast carcinogenesis by inhibiting the anti-tumoral effect of WWOX.

RalB directly triggers invasion downstream Ras by mobilizing the Wave complex.

The two Ral GTPases, RalA and RalB, have crucial roles downstream Ras oncoproteins in human cancers; in particular, RalB is involved in invasion and metastasis. However, therapies targeting Ral signalling are not available yet. By a novel optogenetic approach, we found that light-controlled activation of Ral at plasma-membrane promotes the recruitment of the Wave Regulatory Complex (WRC) via its effector exocyst, with consequent induction of protrusions and invasion. We show that active Ras signals to RalB via two RalGEFs (Guanine nucleotide Exchange Factors), RGL1 and RGL2, to foster invasiveness; RalB contribution appears to be more important than that of MAPK and PI3K pathways. Moreover, on the clinical side, we uncovered a potential role of RalB in human breast cancers by determining that RalB expression at protein level increases in a manner consistent with progression toward metastasis. This work highlights the Ras-RGL1/2-RalB-exocyst-WRC axis as appealing target for novel anticancer strategies.

Coronin 1C promotes triple-negative breast cancer invasiveness through regulation of MT1-MMP traffic and invadopodia function.

Membrane type 1-matrix metalloproteinase (MT1-MMP), a membrane-tethered protease, is key for matrix breakdown during cancer invasion and metastasis. Assembly of branched actin networks by the Arp2/3 complex is required for MT1-MMP traffic and formation of matrix-degradative invadopodia. Contrasting with the well-established role of actin filament branching factor cortactin in invadopodia function during cancer cell invasion, the contribution of coronin-family debranching factors to invadopodia-based matrix remodeling is not known. Here, we investigated the contribution of coronin 1C to the invasive potential of breast cancer cells. We report that expression of coronin 1C is elevated in invasive human breast cancers, correlates positively with MT1-MMP expression in relation with increased metastatic risk and is a new independent prognostic factor in breast cancer. We provide evidence that, akin to cortactin, coronin 1C is required for invadopodia formation and matrix degradation by breast cancer cells lines and for 3D collagen invasion by multicellular spheroids. Using intravital imaging of orthotopic human breast tumor xenografts, we find that coronin 1C accumulates in structures forming in association with collagen fibrils in the tumor microenvironment. Moreover, we establish the role of coronin 1C in the regulation of positioning and trafficking of MT1-MMP-positive endolysosomes. These results identify coronin 1C as a novel player of the multi-faceted mechanism responsible for invadopodia formation, MT1-MMP surface exposure and invasiveness in breast cancer cells.

Medullary Breast Carcinoma, a Triple-Negative Breast Cancer Associated with BCLG Overexpression.

Medullary breast carcinoma (MBC) is a rare subtype of triple-negative breast cancer with specific genomic features within the spectrum of basal-like carcinoma (BLC). In this study of 19 MBCs and 36 non-MBC BLCs, we refined the transcriptomic and genomic knowledge about this entity. Unsupervised and supervised analysis of transcriptomic profiles confirmed that MBC clearly differs from non-MBC BLC, with 92 genes overexpressed and 154 genes underexpressed in MBC compared with non-MBC BLC. Immunity-related pathways are the most differentially represented pathways in MBC compared with non-MBC BLC. The proapoptotic gene BCLG (official name BCL2L14) is by far the most intensely overexpressed gene in MBC. A quantitative RT-PCR validation study conducted in 526 breast tumors corresponding to all molecular subtypes documented the specificity of BCLG overexpression in MBC, which was confirmed at the protein level by immunohistochemistry. We also found that most MBCs belong to the immunomodulatory triple-negative breast cancer subtype. Using pan-genomic analysis, it was found that MBC harbors more losses of heterozygosity than non-MBC BLC. These observations corroborate the notion that MBC remains a distinct entity that could benefit from specific treatment strategies (such as deescalation or targeted therapy) adapted to this rare tumor type.

Morphology and genomic hallmarks of breast tumours developed by ATM deleterious variant carriers.

BACKGROUND: The ataxia telangiectasia mutated (ATM) gene is a moderate-risk breast cancer susceptibility gene; germline loss-of-function variants are found in up to 3% of hereditary breast and ovarian cancer (HBOC) families who undergo genetic testing. So far, no clear histopathological and molecular features of breast tumours occurring in ATM deleterious variant carriers have been described, but identification of an ATM-associated tumour signature may help in patient management. METHODS: To characterise hallmarks of ATM-associated tumours, we performed systematic pathology review of tumours from 21 participants from ataxia-telangiectasia families and 18 participants from HBOC families, as well as copy number profiling on a subset of 23 tumours. Morphology of ATM-associated tumours was compared with that of 599 patients with no BRCA1 and BRCA2 mutations from a hospital-based series, as well as with data from The Cancer Genome Atlas. Absolute copy number and loss of heterozygosity (LOH) profiles were obtained from the OncoScan SNP array. In addition, we performed whole-genome sequencing on four tumours from ATM loss-of-function variant carriers with available frozen material. RESULTS: We found that ATM-associated tumours belong mostly to the luminal B subtype, are tetraploid and show LOH at the ATM locus at 11q22-23. Unlike tumours in which BRCA1 or BRCA2 is inactivated, tumours arising in ATM deleterious variant carriers are not associated with increased large-scale genomic instability as measured by the large-scale state transitions signature. Losses at 13q14.11-q14.3, 17p13.2-p12, 21p11.2-p11.1 and 22q11.23 were observed. Somatic alterations at these loci may therefore represent biomarkers for ATM testing and harbour driver mutations in potentially 'druggable' genes that would allow patients to be directed towards tailored therapeutic strategies. CONCLUSIONS: Although ATM is involved in the DNA damage response, ATM-associated tumours are distinct from BRCA1-associated tumours in terms of morphological characteristics and genomic alterations, and they are also distinguishable from sporadic breast tumours, thus opening up the possibility to identify ATM variant carriers outside the ataxia-telangiectasia disorder and direct them towards effective cancer risk management and therapeutic strategies.

High rate of PIK3CA mutations but no TP53 mutations in low-grade adenosquamous carcinoma of the breast.

AIMS: Low-grade adenosquamous carcinoma of the breast (LGASC) is a rare variant of metaplastic carcinoma characterised by a favourable outcome and histologically composed of glandular and squamous elements in a spindle cell background typically associated with a lymphocytic stromal reaction. Because of its rarity, the immunophenotypic and genetic profile of LGASC has not been sufficiently characterised. The aim of this study was to gain insights into the molecular and phenotypic characteristics of LGASC. METHODS AND RESULTS: We reviewed the clinical and morphological features and detailed the immunohistochemical characteristics of a retrospective series of 13 LGASCs. Targeted sequencing of 50 genes was performed in 10 of 13 cases. Identified mutations were further assessed by Sanger sequencing in a validation series of 11 additional cases. All tumours showed a triple-negative immunophenotype, expressed 'basal' keratins, showed variable levels of epidermal growth factor receptor expression, and did not express androgen receptor. Sequencing analysis of the screening set of LGASCs revealed a high rate (seven of 10 cases) of PIK3CA mutations, whereas no TP53 mutations were found. All PIK3CA mutations were missense mutations located either in exon 20 (n = 6) or in exon 9 (n = 1). The global PIK3CA mutation rate, including the validation series, was 52% (11 of 21 cases). No disease recurrences were observed. [Correction added on 11 June 2018, after first online publication: The percentage of mutation rate was corrected to 52%] CONCLUSIONS: Our results indicate that LGASC of the breast is a low-grade triple-negative breast cancer that harbours a basal-like phenotype with no androgen receptor expression, and shows a high rate of PIK3CA mutations but no TP53 mutations.