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.

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.

E-cadherin mutational landscape and outcomes in breast invasive lobular carcinoma.

Invasive lobular carcinomas (ILC) are characterized by a 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 presence, type or position of CDH1 mutations, E-cadherin expression and clinicopathological features (including outcome) in a retrospective series of 251 primary ILC with 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 two clones (overall concordance: 83.8%, Kappa 0.67): null/focal expression (≤10%) (72.8% of cases for 4A2C7, 83.8% for NCH38), heterogeneous expression (11-89%) (19.2% of cases for 4A2C7, 6.9% for NCH38) and diffuse expression (≥90%) (8% of cases for 4A2C7, 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 non-truncating mutations. Regarding CDH1 mutation location, intracytoplasmic domain mutations correlated with a divergent E-cadherin IHC phenotype between the two antibodies (4A2C7 ≤10% / NCH38 ≥10%). Clinico-pathological correlation analyses found that stromal amount (inversely correlated with tumor cellularity) and TILs were less abundant in ILC with E-cadherin null/focal cases. In addition, CDH1 truncating mutations were associated with radio-histological 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, histological 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 clinico-biological studies dedicated to this tumor type.

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.

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.

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.

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.

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.

Comprehensive clinical and molecular analyses of neuroendocrine carcinomas of the breast.

Neuroendocrine breast carcinomas represent a rare subtype of breast cancer. Their definition, prevalence, and prognosis remain controversial in the literature. The 2012 WHO classification of breast cancer categorizes neuroendocrine carcinomas into three morphologically distinct subtypes: well-differentiated neuroendocrine tumors, poorly differentiated neuroendocrine carcinomas, and invasive breast carcinomas with neuroendocrine differentiation. We aimed to gain insight into the clinical, morphologic, phenotypic, and molecular features of 47 neuroendocrine breast carcinomas. Targeted next-generation sequencing by an AmpliSeq 22 cancer gene hotspot panel and the Prosigna assay were performed on 42/47 and 35/47 cases, respectively. Average age at diagnosis was 69 years. All tumors were estrogen receptor-positive and the large majority expressed progesterone receptor (89%), GATA3 (98%), FOXA1 (96%), and CK8/18 (98%). There was an almost equal distribution of luminal A (52%) and B (48%) carcinomas. Almost half of the cohort (49%) displayed a high risk of recurrence score with the Prosigna test. Patients with a neuroendocrine carcinoma had a shorter disease-free survival compared with those affected by carcinomas of no special type matched for age, size, grade, and estrogen receptor status. No significant differences were observed in terms of overall survival. Stratification of neuroendocrine carcinomas using the 2012 WHO criteria did not reveal statistically significant differences among the distinct categories (well-differentiated neuroendocrine tumors, poorly differentiated neuroendocrine carcinomas, and invasive breast carcinomas with neuroendocrine differentiation), in terms of either progression-free or overall survival. Our targeted sequencing analysis found three cases (7%) harboring a PIK3CA mutation, and in three other cases (7%) TP53 mutations were detected. This study showed that neuroendocrine breast carcinoma is a distinct subtype of luminal carcinoma with a low rate of PIK3CA mutations and with an aggressive clinical behavior. An accurate identification of neuroendocrine differentiation may be useful to better tailor patient adjuvant therapy within luminal carcinomas.

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.

Control of MT1-MMP transport by atypical PKC during breast-cancer progression.

Dissemination of carcinoma cells requires the pericellular degradation of the extracellular matrix, which is mediated by membrane type 1-matrix metalloproteinase (MT1-MMP). In this article, we report a co-up-regulation and colocalization of MT1-MMP and atypical protein kinase C iota (aPKCι) in hormone receptor-negative breast tumors in association with a higher risk of metastasis. Silencing of aPKC in invasive breast-tumor cell lines impaired the delivery of MT1-MMP from late endocytic storage compartments to the surface and inhibited matrix degradation and invasion. We provide evidence that aPKCι, in association with MT1-MMP-containing endosomes, phosphorylates cortactin, which is present in F-actin-rich puncta on MT1-MMP-positive endosomes and regulates cortactin association with the membrane scission protein dynamin-2. Thus, cell line-based observations and clinical data reveal the concerted activity of aPKC, cortactin, and dynamin-2, which control the trafficking of MT1-MMP from late endosome to the plasma membrane and play an important role in the invasive potential of breast-cancer cells.

LIN7A is a major determinant of cell-polarity defects in breast carcinomas.

BACKGROUND: Polarity defects are a hallmark of most carcinomas. Cells from invasive micropapillary carcinomas (IMPCs) of the breast are characterized by a striking cell polarity inversion and represent an interesting model for the analysis of polarity abnormalities. METHODS: In-depth investigation of polarity proteins in 24 IMPCs and a gene expression profiling, comparing IMPC (n = 73) with invasive carcinomas of no special type (ICNST) (n = 51) have been performed. RESULTS: IMPCs showed a profound disorganization of the investigated polarity proteins and revealed major abnormalities in their subcellular localization. Gene expression profiling experiments highlighted a number of deregulated genes in the IMPCs that have a role in apico-basal polarity, adhesion and migration. LIN7A, a Crumbs-complex polarity gene, was one of the most differentially over-expressed genes in the IMPCs. Upon LIN7A over-expression, we observed hyperproliferation, invasion and a complete absence of lumen formation, revealing strong polarity defects. CONCLUSION: This study therefore shows that LIN7A has a crucial role in the polarity abnormalities associated with breast carcinogenesis.

Genetic events in the progression of adenoid cystic carcinoma of the breast to high-grade triple-negative breast cancer.

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.

Genomic landscape of adenoid cystic carcinoma of the breast.

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.

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.

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.

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.