Molecular and Pathologic Characterization of YAP1-Expressing Small Cell Lung Cancer Cell Lines Leads to Reclassification as SMARCA4-Deficient Malignancies.

PURPOSE: The classification of small cell lung cancer (SCLC) into distinct molecular subtypes defined by ASCL1, NEUROD1, POU2F3, or YAP1 (SCLC-A, -N, -P, or -Y) expression, paves the way for a personalized treatment approach. However, the existence of a distinct YAP1-expressing SCLC subtype remains controversial. EXPERIMENTAL DESIGN: To better understand YAP1-expressing SCLC, the mutational landscape of human SCLC cell lines was interrogated to identify pathogenic alterations unique to SCLC-Y. Xenograft tumors, generated from cell lines representing the four SCLC molecular subtypes, were evaluated by a panel of pathologists who routinely diagnose thoracic malignancies. Diagnoses were complemented by transcriptomic analysis of primary tumors and human cell line datasets. Protein expression profiles were validated in patient tumor tissue. RESULTS: Unexpectedly, pathogenic mutations in SMARCA4 were identified in six of eight SCLC-Y cell lines and correlated with reduced SMARCA4 mRNA and protein expression. Pathologist evaluations revealed that SMARCA4-deficient SCLC-Y tumors exhibited features consistent with thoracic SMARCA4-deficient undifferentiated tumors (SMARCA4-UT). Similarly, the transcriptional profile SMARCA4-mutant SCLC-Y lines more closely resembled primary SMARCA4-UT, or SMARCA4-deficient non-small cell carcinoma, than SCLC. Furthermore, SMARCA4-UT patient samples were associated with a YAP1 transcriptional signature and exhibited strong YAP1 protein expression. Together, we found little evidence to support a diagnosis of SCLC for any of the YAP1-expressing cell lines originally used to define the SCLC-Y subtype. CONCLUSIONS: SMARCA4-mutant SCLC-Y cell lines exhibit characteristics consistent with SMARCA4-deficient malignancies rather than SCLC. Our findings suggest that, unlike ASCL1, NEUROD1, and POU2F3, YAP1 is not a subtype defining transcription factor in SCLC. See related commentary by Rekhtman, p. 1708.

Spatial transcriptomics and the anatomical pathologist: Molecular meets morphology.

In recent years anatomical pathology has been revolutionised by the incorporation of molecular findings into routine diagnostic practice, and in some diseases the presence of specific molecular alterations are now essential for diagnosis. Spatial transcriptomics describes a group of technologies that provide up to transcriptome-wide expression profiling while preserving the spatial origin of the data, with many of these technologies able to provide these data using a single tissue section. Spatial transcriptomics allows expression profiling of highly specific areas within a tissue section potentially to subcellular resolution, and allows correlation of expression data with morphology, tissue type and location relative to other structures. While largely still research laboratory-based, several spatial transcriptomics methods have now achieved compatibility with formalin-fixed paraffin-embedded tissue (FFPE), allowing their use in diagnostic tissue samples, and with further development potentially leading to their incorporation in routine anatomical pathology practice. This mini review provides an overview of spatial transcriptomics methods, with an emphasis on platforms compatible with FFPE tissue, approaches to assess the data and potential applications in anatomical pathology practice.

Ultrahigh-precision noble gas isotope analyses reveal pervasive subsurface fractionation in hydrothermal systems.

Mantle-derived noble gases in volcanic gases are powerful tracers of terrestrial volatile evolution, as they contain mixtures of both primordial (from Earth's accretion) and secondary (e.g., radiogenic) isotope signals that characterize the composition of deep Earth. However, volcanic gases emitted through subaerial hydrothermal systems also contain contributions from shallow reservoirs (groundwater, crust, atmosphere). Deconvolving deep and shallow source signals is critical for robust interpretations of mantle-derived signals. Here, we use a novel dynamic mass spectrometry technique to measure argon, krypton, and xenon isotopes in volcanic gas with ultrahigh precision. Data from Iceland, Germany, United States (Yellowstone, Salton Sea), Costa Rica, and Chile show that subsurface isotope fractionation within hydrothermal systems is a globally pervasive and previously unrecognized process causing substantial nonradiogenic Ar-Kr-Xe isotope variations. Quantitatively accounting for this process is vital for accurately interpreting mantle-derived volatile (e.g., noble gas and nitrogen) signals, with profound implications for our understanding of terrestrial volatile evolution.

Temporal variation of planetary iron as a driver of evolution.

Iron is an irreplaceable component of proteins and enzyme systems required for life. This need for iron is a well-characterized evolutionary mechanism for genetic selection. However, there is limited consideration of how iron bioavailability, initially determined by planetary accretion but fluctuating considerably at global scale over geological time frames, has shaped the biosphere. We describe influences of iron on planetary habitability from formation events >4 Gya and initiation of biochemistry from geochemistry through oxygenation of the atmosphere to current host-pathogen dynamics. By determining the iron and transition element distribution within the terrestrial planets, planetary core formation is a constraint on both the crustal composition and the longevity of surface water, hence a planet's habitability. As such, stellar compositions, combined with metallic core-mass fraction, may be an observable characteristic of exoplanets that relates to their ability to support life. On Earth, the stepwise rise of atmospheric oxygen effectively removed gigatons of soluble ferrous iron from habitats, generating evolutionary pressures. Phagocytic, infectious, and symbiotic behaviors, dating from around the Great Oxygenation Event, refocused iron acquisition onto biotic sources, while eukaryotic multicellularity allows iron recycling within an organism. These developments allow life to more efficiently utilize a scarce but vital nutrient. Initiation of terrestrial life benefitted from the biochemical properties of abundant mantle/crustal iron, but the subsequent loss of iron bioavailability may have been an equally important driver of compensatory diversity. This latter concept may have relevance for the predicted future increase in iron deficiency across the food chain caused by elevated atmospheric CO2.

SP142 PD-L1 Scoring Shows High Interobserver and Intraobserver Agreement in Triple-negative Breast Carcinoma But Overall Low Percentage Agreement With Other PD-L1 Clones SP263 and 22C3.

SP142 programmed cell death ligand 1 (PD-L1) status predicts response to atezolizumab in triple-negative breast carcinoma (TNBC). Prevalence of VENTANA PD-L1 (SP142) Assay positivity, concordance with the VENTANA PD-L1 (SP263) Assay and Dako PD-L1 IHC 22C3 pharmDx assay, and association with clinicopathologic features were assessed in 447 TNBCs. SP142 PD-L1 intraobserver and interobserver agreement was investigated in a subset of 60 TNBCs, with scores enriched around the 1% cutoff. The effect of a 1-hour training video on pretraining and posttraining scores was ascertained. At a 1% cutoff, 34.2% of tumors were SP142 PD-L1 positive. SP142 PD-L1 positivity was significantly associated with tumor-infiltrating lymphocytes (P <0.01), and node negativity (P=0.02), but not with tumor grade (P=0.35), tumor size (P=0.58), or BRCA mutation (P=0.53). Overall percentage agreement (OPA) for intraobserver and interobserver agreement was 95.0% and 93.7%, respectively, among 5 pathologists trained in TNBC SP142 PD-L1 scoring. In 5 TNBC SP142 PD-L1-naive pathologists, significantly higher OPA to the reference score was achieved after video training (posttraining OPA 85.7%, pretraining OPA 81.5%, P<0.05). PD-L1 status at a 1% cutoff was assessed by SP142 and SP263 in 420 cases, and by SP142 and 22C3 in 423 cases, with OPA of 88.1% and 85.8%, respectively. The VENTANA PD-L1 (SP142) Assay is reproducible for classifying TNBC PD-L1 status by trained observers; however, it is not analytically equivalent to the VENTANA PD-L1 (SP263) Assay and Dako PD-L1 IHC 22C3 pharmDx assay.

A 4,565-My-old andesite from an extinct chondritic protoplanet.

The age of iron meteorites implies that accretion of protoplanets began during the first millions of years of the solar system. Due to the heat generated by 26Al decay, many early protoplanets were fully differentiated with an igneous crust produced during the cooling of a magma ocean and the segregation at depth of a metallic core. The formation and nature of the primordial crust generated during the early stages of melting is poorly understood, due in part to the scarcity of available samples. The newly discovered meteorite Erg Chech 002 (EC 002) originates from one such primitive igneous crust and has an andesite bulk composition. It derives from the partial melting of a noncarbonaceous chondritic reservoir, with no depletion in alkalis relative to the Sun's photosphere and at a high degree of melting of around 25%. Moreover, EC 002 is, to date, the oldest known piece of an igneous crust with a 26Al-26Mg crystallization age of 4,565.0 million years (My). Partial melting took place at 1,220 °C up to several hundred kyr before, implying an accretion of the EC 002 parent body ca. 4,566 My ago. Protoplanets covered by andesitic crusts were probably frequent. However, no asteroid shares the spectral features of EC 002, indicating that almost all of these bodies have disappeared, either because they went on to form the building blocks of larger bodies or planets or were simply destroyed.

A MXI1-NUTM1 fusion protein with MYC-like activity suggests a novel oncogenic mechanism in a subset of NUTM1-rearranged tumors.

Most NUTM1-rearranged neoplasms (NRNs) have fusions between NUTM1 and BRD (bromodomain-containing) family members and are termed NUT carcinomas (NCs) because they show some squamous differentiation. However, some NRNs are associated with fusions between NUTM1 and members of the MAD (MAX dimerization) gene family of MYC antagonists. Here we describe a small round cell malignancy from the gastro-esophageal junction with a previously unreported fusion between NUTM1 and the MAD family member MXI1. In contrast to NCs, the MXI1-NUTM1 tumor did not show squamous differentiation and did not express MYC, TP63 or SOX2, genes known to be targets of BRD-NUTM1 proteins and critical for NC oncogenesis. Transcriptome analysis showed paradoxical enrichment of MYC target genes in the MXI1-NUTM1 tumor despite the lack of MYC expression. When expressed in vitro MXI1-NUTM1 partially phenocopied MYC, enhancing cell proliferation and cooperating with oncogenic HRAS to produce anchorage-independent cell growth. These data provide evidence that MAD family members, which are normally repressors of MYC activity, can be converted into MYC-like mimics by fusion to NUTM1. The pathological features and novel oncogenic mechanism of the MXI1-NUTM1 tumor show that identification of NUTM1 fusion partners can be important for accurate diagnostic classification of some NRN subtypes, and potentially may guide therapeutic options.

Immune molecular profiling of a multiresistant primary prostate cancer with a neuroendocrine-like phenotype: a case report.

BACKGROUND: Understanding the drivers of recurrence in aggressive prostate cancer requires detailed molecular and genomic understanding in order to aid therapeutic interventions. We provide here a case report of histological, transcriptional, proteomic, immunological, and genomic features in a longitudinal study of multiple biopsies from diagnosis, through treatment, and subsequent recurrence. CASE PRESENTATION: Here we present a case study of a male in 70 s with high-grade clinically-localised acinar adenocarcinoma treated with definitive hormone therapy and radiotherapy. The patient progressed rapidly with rising PSA and succumbed without metastasis 52 months after diagnosis. We identified the expression of canonical histological markers of neuroendocrine PC (NEPC) including synaptophysin, neuron-specific enolase and thyroid transcription factor 1, as well as intact AR expression, in the recurrent disease only. The resistant disease was also marked by an extremely low immune infiltrate, extensive genomic chromosomal aberrations, and overactivity in molecular hallmarks of NEPC disease including Aurora kinase and E2F, as well as novel alterations in the cMYB pathway. We also observed that responses to both primary treatments (high dose-rate brachytherapy and androgen deprivation therapies) were consistent with known optimal responses-ruling out treatment inefficacy as a factor in relapse. CONCLUSIONS: These data provide novel insights into a case of locally recurrent aggressive prostate cancer harbouring NEPC pathology, in the absence of detected metastasis.

High dose-rate brachytherapy of localized prostate cancer converts tumors from cold to hot.

BACKGROUND: Prostate cancer (PCa) has a profoundly immunosuppressive microenvironment and is commonly immune excluded with few infiltrative lymphocytes and low levels of immune activation. High-dose radiation has been demonstrated to stimulate the immune system in various human solid tumors. We hypothesized that localized radiation therapy, in the form of high dose-rate brachytherapy (HDRBT), would overcome immune suppression in PCa. METHODS: To investigate whether HDRBT altered prostate immune context, we analyzed preradiation versus postradiation human tissue from a cohort of 24 patients with localized PCa that received HDRBT as primary treatment (RadBank cohort). We performed Nanostring immune gene expression profiling, digital spatial profiling, and high-throughput immune cell multiplex immunohistochemistry analysis. We also resolved tumor and nontumor zones in spatial and bioinformatic analyses to explore the immunological response. RESULTS: Nanostring immune profiling revealed numerous immune checkpoint molecules (eg, B7-H3, CTLA4, PDL1, and PDL2) and TGFß levels were increased in response to HDRBT. We used a published 16-gene tumor inflammation signature (TIS) to divide tumors into distinct immune activation states (high:hot, intermediate and low:cold) and showed that most localized PCa are cold tumors pre-HDRBT. Crucially, HDRBT converted 80% of these 'cold'-phenotype tumors into an 'intermediate' or 'hot' class. We used digital spatial profiling to show these HDRBT-induced changes in prostate TIS scores were derived from the nontumor regions. Furthermore, these changes in TIS were also associated with pervasive changes in immune cell density and spatial relationships-in particular, between T cell subsets and antigen presenting cells. We identified an increased density of CD4+ FOXP3+ T cells, CD68+ macrophages and CD68+ CD11c+ dendritic cells in response to HDRBT. The only subset change specific to tumor zones was PDL1- macrophages. While these immune responses were heterogeneous, HDRBT induced significant changes in immune cell associations, including a gained T cell and HMWCK+ PDL1+ interaction in tumor zones. CONCLUSION: In conclusion, we showed HDRBT converted "cold" prostate tumors into more immunologically activated "hot" tissues, with accompanying spatially organized immune infiltrates and signaling changes. Understanding and potentially harnessing these changes will have widespread implications for the future treatment of localized PCa, including rational use of combination radio-immunotherapy.

Identification of chondritic krypton and xenon in Yellowstone gases and the timing of terrestrial volatile accretion.

Identifying the origin of noble gases in Earth's mantle can provide crucial constraints on the source and timing of volatile (C, N, H2O, noble gases, etc.) delivery to Earth. It remains unclear whether the early Earth was able to directly capture and retain volatiles throughout accretion or whether it accreted anhydrously and subsequently acquired volatiles through later additions of chondritic material. Here, we report high-precision noble gas isotopic data from volcanic gases emanating from, in and around, the Yellowstone caldera (Wyoming, United States). We show that the He and Ne isotopic and elemental signatures of the Yellowstone gas requires an input from an undegassed mantle plume. Coupled with the distinct ratio of 129Xe to primordial Xe isotopes in Yellowstone compared with mid-ocean ridge basalt (MORB) samples, this confirms that the deep plume and shallow MORB mantles have remained distinct from one another for the majority of Earth's history. Krypton and xenon isotopes in the Yellowstone mantle plume are found to be chondritic in origin, similar to the MORB source mantle. This is in contrast with the origin of neon in the mantle, which exhibits an isotopic dichotomy between solar plume and chondritic MORB mantle sources. The co-occurrence of solar and chondritic noble gases in the deep mantle is thought to reflect the heterogeneous nature of Earth's volatile accretion during the lifetime of the protosolar nebula. It notably implies that the Earth was able to retain its chondritic volatiles since its earliest stages of accretion, and not only through late additions.

RAF1 rearrangements are common in pancreatic acinar cell carcinomas.

There is now evidence that gene fusions activating the MAPK pathway are relatively common in pancreatic acinar cell carcinoma with potentially actionable BRAF or RET fusions being found in ~30%. We sought to investigate the incidence of RAF1 fusions in pancreatic malignancies with acinar cell differentiation. FISH testing for RAF1 was undertaken on 30 tumors comprising 25 'pure' acinar cell carcinomas, 2 mixed pancreatic acinar-neuroendocrine carcinomas, 1 mixed acinar cell-low grade neuroendocrine tumor and 2 pancreatoblastomas. RAF1 rearrangements were identified in 5 cases and confirmed by DNA and RNA sequencing to represent oncogenic fusions (GATM-RAF1, GOLGA4-RAF1, PDZRN3-RAF1, HERPUD1-RAF1 and TRIM33-RAF1) and to be mutually exclusive with BRAF and RET fusions, as well as KRAS mutations. Large genome-wide copy number changes were common and included 1q gain and/or 1p loss in all five RAF1 FISH-positive acinar cell carcinomas. RAF1 expression by immunohistochemistry was found in 3 of 5 (60%) of fusion-positive cases and no FISH-negative cases. Phospho-ERK1/2 expression was found in 4 of 5 RAF1-fusion-positive cases. Expression of both RAF1 and phospho-ERK1/2 was heterogeneous and often only detected at the tumor-stroma interface, thus limiting their clinical utility. We conclude that RAF1 gene rearrangements are relatively common in pancreatic acinar cell carcinomas (14.3% to 18.5% of cases) and can be effectively identified by FISH with follow up molecular testing. The combined results of several studies now indicate that BRAF, RET or RAF1 fusions occur in between one third and one-half of these tumors but are extremely rare in other pancreatic malignancies. As these fusions are potentially actionable with currently available therapies, a strong argument can be made to perform FISH or molecular testing on all pancreatic acinar cell carcinomas.

The genetic architecture of breast papillary lesions as a predictor of progression to carcinoma.

Intraductal papillomas (IDP) are challenging breast findings because of their variable risk of progression to malignancy. The molecular events driving IDP development and genomic features of malignant progression are poorly understood. In this study, genome-wide CNA and/or targeted mutation analysis was performed on 44 cases of IDP, of which 20 cases had coexisting ductal carcinoma in situ (DCIS), papillary DCIS or invasive ductal carcinoma (IDC). CNA were rare in pure IDP, but 69% carried an activating PIK3CA mutation. Among the synchronous IDP cases, 55% (11/20) were clonally related to the synchronous DCIS and/or IDC, only one of which had papillary histology. In contrast to pure IDP, PIK3CA mutations were absent from clonal cases. CNAs in any of chromosomes 1, 16 or 11 were significantly enriched in clonal IDP lesions compared to pure and non-clonal IDP. The observation that 55% of IDP are clonal to DCIS/IDC indicates that IDP can be a direct precursor for breast carcinoma, not limited to the papillary type. The absence of PIK3CA mutations and presence of CNAs in IDP could be used clinically to identify patients at high risk of progression to carcinoma.

Adequate tumour cellularity is essential for accurate PD-L1 immunohistochemistry assessment on cytology cell-block specimens.

OBJECTIVES: PD-L1 immunohistochemistry (IHC) is an essential predictive biomarker for patients with non-small cell lung cancer (NSCLC), required to inform treatment decisions regarding anti-PD-1 immune checkpoint inhibitor therapy. This study aims to investigate the concordance between PD-L1 IHC assessed on NSCLC cytology and histology specimens and to determine the impactce of tumour cellularity. METHODS: Matched cytology and histology NSCLC specimens were retrieved from the archives of the Royal Melbourne Hospital and the Royal Prince Alfred Hospital. PD-L1 IHC was performed concurrently on both specimens at the Peter MacCallum Cancer Centre using the SP263 assay kit on the Ventana Benchmark Ultra staining platform and scored by two experienced pathologists. RESULTS: Overall agreement between matched cytology and histology specimens was good (intraclass correlation coefficient = 0.653, n = 58); however, markedly increased when the analysis was limited to cell-blocks with >100 tumour cells (intraclass correlation coefficient = 0.957, n = 29). Specificity at both 1% and 50% cut-offs was high regardless of cellularity; however, sensitivity decreased in samples with <100 tumour cells. CONCLUSIONS: PD-L1 IHC on cytology cell-block specimens in NSCLC is an acceptable alternative to histological specimens, provided adequate tumour cells are present. Clinicians and pathologists should be mindful of the risk of false negative PD-L1 IHC in samples with low tumour cellularity, to avoid excluding patients from potentially beneficial treatment.

Preoperative ß-Blockade with Propranolol Reduces Biomarkers of Metastasis in Breast Cancer: A Phase II Randomized Trial.

PURPOSE: The majority of deaths from breast cancer occur following the development of metastatic disease, a process inhibited by ß-blockers in preclinical studies. This phase II randomized controlled trial evaluated the effect of preoperative ß-blockade with propranolol on biomarkers of metastatic potential and the immune cell profile within the primary tumor of patients with breast cancer. PATIENTS AND METHODS: In this triple-blind placebo-controlled clinical trial, 60 patients were randomly assigned to receive an escalating dose of oral propranolol (n = 30; 80-160 mg daily) or placebo (n = 30) for 7 days prior to surgery. The primary endpoint investigated the effect of propranolol on prometastatic and proinflammatory gene expression within the primary tumor. RESULTS: Propranolol downregulated primary tumor expression of mesenchymal genes (P = 0.002) without affecting epithelial gene expression (P = 0.21). Bioinformatic analyses implicated downregulation of Snail/Slug (P = 0.03), NF-κB/Rel (P < 0.01), and AP-1 (P < 0.01) transcription factors in structuring the observed transcriptome alterations, and identified changes in intratumoral neutrophil, natural killer cell, and dendritic cell recruitment (all P < 0.01). Patients with clinical evidence of drug response (lowered heart rate and blood pressure) demonstrated elevated tumor infiltration of CD68+ macrophages and CD8+ T cells. CONCLUSIONS: One week of ß-blockade with propranolol reduced intratumoral mesenchymal polarization and promoted immune cell infiltration in early-stage surgically-resectable breast cancer. These results show that ß-blockade reduces biomarkers associated with metastatic potential, and support the need for larger phase III clinical trials powered to detect the impact of ß-blockade on cancer recurrence and survival.See related commentary by Blaes et al., p. 1781.

Atypical ductal hyperplasia is a multipotent precursor of breast carcinoma.

The current model for breast cancer progression proposes independent 'low grade (LG)-like' and 'high grade (HG)-like' pathways but lacks a known precursor to HG cancer. We applied low-coverage whole-genome sequencing to atypical ductal hyperplasia (ADH) with and without carcinoma to shed light on breast cancer progression. Fourteen out of twenty isolated ADH cases harboured at least one copy number alteration (CNA), but had fewer aberrations than LG or HG ductal carcinoma in situ (DCIS). ADH carried more HG-like CNA than LG DCIS (e.g. 8q gain). Correspondingly, 64% (7/11) of ADH cases with synchronous HG carcinoma were clonally related, similar to LG carcinoma (67%, 6/9). This study represents a significant shift in our understanding of breast cancer progression, with ADH as a common precursor lesion to the independent 'low grade-like' and 'high grade-like' pathways. These data suggest that ADH can be a precursor of HG breast cancer and that LG and HG carcinomas can evolve from a similar ancestor lesion. We propose that although LG DCIS may be committed to a LG molecular pathway, ADH may remain multipotent, progressing to either LG or HG carcinoma. This multipotent nature suggests that some ADH cases could be more clinically significant than LG DCIS, requiring biomarkers for personalising management. Copyright © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Profound MEK inhibitor response in a cutaneous melanoma harboring a GOLGA4-RAF1 fusion.

BRAF and CRAF are critical components of the MAPK signaling pathway which is activated in many cancer types. In approximately 1% of melanomas, BRAF or CRAF are activated through structural arrangements. We describe here a metastatic melanoma with a GOLGA4-RAF1 fusion and pathogenic variants in CTNNB1 and CDKN2A. Anti-CTLA4/anti-PD1 combination immunotherapy failed to control tumor progression. In the absence of other actionable variants the patient was administered MEK inhibitor therapy on the basis of its potential action against RAF1 fusions. This resulted in a profound and clinically significant response. We demonstrated that GOLGA4-RAF1 expression was associated with ERK activation, elevated expression of the RAS/RAF downstream co-effector ETV5, and a high Ki67 index. These findings provide a rationale for the dramatic response to targeted therapy. This study shows that thorough molecular characterization of treatment-resistant cancers can identify therapeutic targets and personalize management, leading to improved patient outcomes.

CCL27/CCL28-CCR10 Chemokine Signaling Mediates Migration of Lymphatic Endothelial Cells.

Metastasis via the lymphatic vasculature is an important step in cancer progression. The formation of new lymphatic vessels (lymphangiogenesis), or remodeling of existing lymphatics, is thought to facilitate the entry and transport of tumor cells into lymphatic vessels and on to distant organs. The migration of lymphatic endothelial cells (LEC) toward guidance cues is critical for lymphangiogenesis. While chemokines are known to provide directional navigation for migrating immune cells, their role in mediating LEC migration during tumor-associated lymphangiogenesis is not well defined. Here, we undertook gene profiling studies to identify chemokine-chemokine receptor pairs that are involved in tumor lymphangiogenesis associated with lymph node metastasis. CCL27 and CCL28 were expressed in tumor cells with metastatic potential, while their cognate receptor, CCR10, was expressed by LECs and upregulated by the lymphangiogenic growth factor VEGFD and the proinflammatory cytokine TNFα. Migration assays demonstrated that LECs are attracted to both CCL27 and CCL28 in a CCR10-dependent manner, while abnormal lymphatic vessel patterning in CCR10-deficient mice confirmed the significant role of CCR10 in lymphatic patterning. In vivo analyses showed that LECs are recruited to a CCL27 or CCL28 source, while VEGFD was required in combination with these chemokines to enable formation of coherent lymphatic vessels. Moreover, tumor xenograft experiments demonstrated that even though CCL27 expression by tumors enhanced LEC recruitment, the ability to metastasize was dependent on the expression of VEGFD. These studies demonstrate that CCL27 and CCL28 signaling through CCR10 may cooperate with inflammatory mediators and VEGFD during tumor lymphangiogenesis. SIGNIFICANCE: The study shows that the remodeling of lymphatic vessels in cancer is influenced by CCL27 and CCL28 chemokines, which may provide a future target to modulate metastatic spread.

Frequent activating STAT3 mutations and novel recurrent genomic abnormalities detected in breast implant-associated anaplastic large cell lymphoma.

Breast implant-associated anaplastic large cell lymphoma (BIA-ALCL) is a rare form of T-cell lymphoma that occurs after implantation of breast prostheses. We performed comprehensive next generation sequencing based genomic characterization of 11 cases of BIA-ALCL including sequence variant detection on 180 genes frequently mutated in haematological malignancy, genome-wide copy number assessment, structural variant detection involving the T-cell receptor loci and TRB deep-sequencing. We observed sequence variants leading to JAK/STAT activation in 10 out of 11 patients. We also observed germline TP53 mutations in two cases. In addition we detected a recurrent copy number loss involving RPL5 as well as copy number amplifications involving TNFRSF11A [RANK] (in 2 cases), MYC, P2RX7, TMEM119 and PDGFRA. In summary, our comprehensive genomic characterisation of 11 cases of BIA-ALCL has provided insight into potential pathobiological mechanisms (JAK/STAT, MYC and TP53) as well as identifying targets for future therapeutic intervention (TNFRSF11A, PDGFRA) in this rare entity.

Publisher Correction: Single-cell profiling of breast cancer T cells reveals a tissue-resident memory subset associated with improved prognosis.

In the version of this article originally published, the institution in affiliation 10 was missing. Affiliation 10 was originally listed as Department of Surgery, Royal Melbourne Hospital and Royal Womens' Hospital, Melbourne, Victoria, Australia. It should have been Department of Surgery, Royal Melbourne Hospital and Royal Womens' Hospital, University of Melbourne, Melbourne, Victoria, Australia. The error has been corrected in the HTML and PDF versions of this article.

Single-cell profiling of breast cancer T cells reveals a tissue-resident memory subset associated with improved prognosis.

The quantity of tumor-infiltrating lymphocytes (TILs) in breast cancer (BC) is a robust prognostic factor for improved patient survival, particularly in triple-negative and HER2-overexpressing BC subtypes1. Although T cells are the predominant TIL population2, the relationship between quantitative and qualitative differences in T cell subpopulations and patient prognosis remains unknown. We performed single-cell RNA sequencing (scRNA-seq) of 6,311 T cells isolated from human BCs and show that significant heterogeneity exists in the infiltrating T cell population. We demonstrate that BCs with a high number of TILs contained CD8+ T cells with features of tissue-resident memory T (TRM) cell differentiation and that these CD8+ TRM cells expressed high levels of immune checkpoint molecules and effector proteins. A CD8+ TRM gene signature developed from the scRNA-seq data was significantly associated with improved patient survival in early-stage triple-negative breast cancer (TNBC) and provided better prognostication than CD8 expression alone. Our data suggest that CD8+ TRM cells contribute to BC immunosurveillance and are the key targets of modulation by immune checkpoint inhibition. Further understanding of the development, maintenance and regulation of TRM cells will be crucial for successful immunotherapeutic development in BC.