The Genetic Landscape of Primary Breast Marginal Zone Lymphoma Identifies a Mutational-driven Disease With Similarities to Ocular Adnexal Lymphoma.

Extranodal marginal zone lymphomas (eMZL) can occur in any organ and site of the body. Recent research has shown that they differ from organ to organ in terms of their mutational profile. In this study, we investigated a cohort of primary breast marginal zone lymphomas (PBMZL) to get a better insight into their morphologic and molecular profile. A cohort of 15 cases (14 female and 1 male) was characterized by immunohistochemistry (IHC) for 19 markers, fluorescence in situ hybridization (FISH), and high throughput sequencing (HTS) using a lymphoma panel comprising 172 genes. In addition, PCR for the specific detection of Borrelia spp. and metagenomics whole genome sequencing were performed for infectious agent profiling. Follicular colonization was observed in most cases, while lymphoepithelial lesions, though seen in many cases, were not striking. All 15 cases were negative for CD5, CD11c, and CD21 and positive for BCL2 and pan B-cell markers. There were no cases with BCL2, BCL10, IRF4, MALT1, or MYC translocation; only 1 had a BCL6 rearrangement. HTS highlighted TNFAIP3 (n=4), KMT2D (n=2), and SPEN (n=2) as the most frequently mutated genes. There were no Borrelia spp., and no other pathogens detected in our cohort. One patient had a clinical history of erythema chronicum migrans affecting the same breast. PBMZL is a mutation-driven disease rather than fusion-driven. It exhibits mutations in genes encoding components affecting the NF-κB pathway, chromatin modifier-encoding genes, and NOTCH pathway-related genes. Its mutational profile shares similarities with ocular adnexal and nodal MZL.

Single-cell Analysis Reveals Inter- and Intratumour Heterogeneity in Metastatic Breast Cancer.

Metastasis is the leading cause of cancer-related deaths of breast cancer patients. Some cancer cells in a tumour go through successive steps, referred to as the metastatic cascade, and give rise to metastases at a distant site. We know that the plasticity and heterogeneity of cancer cells play critical roles in metastasis but the precise underlying molecular mechanisms remain elusive. Here we aimed to identify molecular mechanisms of metastasis during colonization, one of the most important yet poorly understood steps of the cascade. We performed single-cell RNA-Seq (scRNA-Seq) on tumours and matched lung macrometastases of patient-derived xenografts of breast cancer. After correcting for confounding factors such as the cell cycle and the percentage of detected genes (PDG), we identified cells in three states in both tumours and metastases. Gene-set enrichment analysis revealed biological processes specific to proliferation and invasion in two states. Our findings suggest that these states are a balance between epithelial-to-mesenchymal (EMT) and mesenchymal-to-epithelial transitions (MET) traits that results in so-called partial EMT phenotypes. Analysis of the top differentially expressed genes (DEGs) between these cell states revealed a common set of partial EMT transcription factors (TFs) controlling gene expression, including ZNF750, OVOL2, TP63, TFAP2C and HEY2. Our data suggest that the TFs related to EMT delineate different cell states in tumours and metastases. The results highlight the marked interpatient heterogeneity of breast cancer but identify common features of single cells from five models of metastatic breast cancer.

Bone marrow haematopoiesis in patients with COVID-19.

AIMS: Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) infection broadly affects organ homeostasis, including the haematopoietic system. Autopsy studies are a crucial tool for investigation of organ-specific pathologies. Here we perform an in-depth analysis of the impact of severe coronavirus disease 2019 (COVID-19) on bone marrow haematopoiesis in correlation with clinical and laboratory parameters. METHODS AND RESULTS: Twenty-eight autopsy cases and five controls from two academic centres were included in the study. We performed a comprehensive analysis of bone marrow pathology and microenvironment features with clinical and laboratory parameters and assessed SARS-CoV-2 infection of the bone marrow by quantitative polymerase chain reaction (qPCR) analysis. In COVID-19 patients, bone marrow specimens showed a left-shifted myelopoiesis (19 of 28, 64%), increased myeloid-erythroid ratio (eight of 28, 28%), increased megakaryopoiesis (six of 28, 21%) and lymphocytosis (four of 28, 14%). Strikingly, a high proportion of COVID-19 specimens showed erythrophagocytosis (15 of 28, 54%) and the presence of siderophages (11 of 15, 73%) compared to control cases (none of five, 0%). Clinically, erythrophagocytosis correlated with lower haemoglobin levels and was more frequently observed in patients from the second wave. Analysis of the immune environment showed a strong increase in CD68+ macrophages (16 of 28, 57%) and a borderline lymphocytosis (five of 28, 18%). The stromal microenvironment showed oedema (two of 28, 7%) and severe capillary congestion (one of 28, 4%) in isolated cases. No stromal fibrosis or microvascular thrombosis was found. While all cases had confirmed positive testing of SARS-CoV-2 in the respiratory system, SARS-CoV-2 was not detected in the bone marrow by high-sensitivity PCR, suggesting that SARS-CoV-2 does not commonly replicate in the haematopoietic microenvironment. CONCLUSIONS: SARS-CoV-2 infection indirectly impacts the haematological compartment and the bone marrow immune environment. Erythrophagocytosis is frequent and associated with lower haemoglobin levels in patients with severe COVID-19.

Nicotinamide N-methyltransferase sustains a core epigenetic program that promotes metastatic colonization in breast cancer.

Metastatic colonization of distant organs accounts for over 90% of deaths related to solid cancers, yet the molecular determinants of metastasis remain poorly understood. Here, we unveil a mechanism of colonization in the aggressive basal-like subtype of breast cancer that is driven by the NAD+ metabolic enzyme nicotinamide N-methyltransferase (NNMT). We demonstrate that NNMT imprints a basal genetic program into cancer cells, enhancing their plasticity. In line, NNMT expression is associated with poor clinical outcomes in patients with breast cancer. Accordingly, ablation of NNMT dramatically suppresses metastasis formation in pre-clinical mouse models. Mechanistically, NNMT depletion results in a methyl overflow that increases histone H3K9 trimethylation (H3K9me3) and DNA methylation at the promoters of PR/SET Domain-5 (PRDM5) and extracellular matrix-related genes. PRDM5 emerged in this study as a pro-metastatic gene acting via induction of cancer-cell intrinsic transcription of collagens. Depletion of PRDM5 in tumor cells decreases COL1A1 deposition and impairs metastatic colonization of the lungs. These findings reveal a critical activity of the NNMT-PRDM5-COL1A1 axis for cancer cell plasticity and metastasis in basal-like breast cancer.

PI3K inhibition circumvents resistance to SHP2 blockade in metastatic triple-negative breast cancer.

The protein tyrosine phosphatase SHP2 activates oncogenic pathways downstream of most receptor tyrosine kinases (RTK) and has been implicated in various cancer types, including the highly aggressive subtype of triple-negative breast cancer (TNBC). Although allosteric inhibitors of SHP2 have been developed and are currently being evaluated in clinical trials, neither the mechanisms of the resistance to these agents, nor the means to circumvent such resistance have been clearly defined. The PI3K signaling pathway is also hyperactivated in breast cancer and contributes to resistance to anticancer therapies. When PI3K is inhibited, resistance also develops for example via activation of RTKs. We therefore assessed the effect of targeting PI3K and SHP2 alone or in combination in preclinical models of metastatic TNBC. In addition to the beneficial inhibitory effects of SHP2 alone, dual PI3K/SHP2 treatment decreased primary tumor growth synergistically, blocked the formation of lung metastases, and increased survival in preclinical models. Mechanistically, transcriptome and phospho-proteome analyses revealed that resistance to SHP2 inhibition is mediated by PDGFRß-evoked activation of PI3K signaling. Altogether, our data provide a rationale for co-targeting of SHP2 and PI3K in metastatic TNBC.

Unbiased screen for pathogens in human paraffin-embedded tissue samples by whole genome sequencing and metagenomics.

Identification of bacterial pathogens in formalin fixed, paraffin embedded (FFPE) tissue samples is limited to targeted and resource-intensive methods such as sequential PCR analyses. To enable unbiased screening for pathogens in FFPE tissue samples, we established a whole genome sequencing (WGS) method that combines shotgun sequencing and metagenomics for taxonomic identification of bacterial pathogens after subtraction of human genomic reads. To validate the assay, we analyzed more than 100 samples of known composition as well as FFPE lung autopsy tissues with and without histological signs of infections. Metagenomics analysis confirmed the pathogenic species that were previously identified by species-specific PCR in 62% of samples, showing that metagenomics is less sensitive than species-specific PCR. On the other hand, metagenomics analysis identified pathogens in samples, which had been tested negative for multiple common microorganisms and showed histological signs of infection. This highlights the ability of this assay to screen for unknown pathogens and detect multi-microbial infections which is not possible by histomorphology and species-specific PCR alone.

Glucocorticoids promote breast cancer metastasis.

Diversity within or between tumours and metastases (known as intra-patient tumour heterogeneity) that develops during disease progression is a serious hurdle for therapy1-3. Metastasis is the fatal hallmark of cancer and the mechanisms of colonization, the most complex step in the metastatic cascade4, remain poorly defined. A clearer understanding of the cellular and molecular processes that underlie both intra-patient tumour heterogeneity and metastasis is crucial for the success of personalized cancer therapy. Here, using transcriptional profiling of tumours and matched metastases in patient-derived xenograft models in mice, we show cancer-site-specific phenotypes and increased glucocorticoid receptor activity in distant metastases. The glucocorticoid receptor mediates the effects of stress hormones, and of synthetic derivatives of these hormones that are used widely in the clinic as anti-inflammatory and immunosuppressive agents. We show that the increase in stress hormones during breast cancer progression results in the activation of the glucocorticoid receptor at distant metastatic sites, increased colonization and reduced survival. Our transcriptomics, proteomics and phospho-proteomics studies implicate the glucocorticoid receptor in the activation of multiple processes in metastasis and in the increased expression of kinase ROR1, both of which correlate with reduced survival. The ablation of ROR1 reduced metastatic outgrowth and prolonged survival in preclinical models. Our results indicate that the activation of the glucocorticoid receptor increases heterogeneity and metastasis, which suggests that caution is needed when using glucocorticoids to treat patients with breast cancer who have developed cancer-related complications.