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Background: Heterozygous hotspot mutations in the RNA splicing factor SF3B1, occur in 3% of unselected breast cancers and are associated with oestrogen receptor (ER+) breast cancer (BC) where they are enriched in metastatic disease and are associated with a poor clinical outcome. SF3B1 mutations drive distinct signatures of alternative splicing through cryptic 3’ splice site selection leading to global transcriptomic and proteomic changes. The functional consequences of the mis-splicing events and resultant genetic vulnerabilities are poorly understood and precision medicine approaches that exploit these characteristics are not clinically available (Table 1). Methods: To understand the role of SF3B1 mutations in ER+ BC, we generated a series of SF3B1 mutant (SF3B1MUT) isogenic cell lines which were characterised using RNA-sequencing and high content mass-spectrometry proteomic profiling. SF3B1 interactome analysis was also performed using immunoprecipitation of SF3B1 followed by mass-spectrometry. The molecular consequences of aberrant splicing were investigated using a targeted screening approach of 280 genes predicted to be alternatively spliced in SF3B1MUT BC, while high-throughput drug screens were used to identify novel therapeutic options for patients with SF3B1MUT breast cancer using isogenic cells. Hits were validated in vitro and in vivo using cell line and patient derived xenografts. Results: Transcriptomic and proteomic profiling of SF3B1MUT cells identified global alternative 3’ splice site selection and subsequent proteomic changes induced by the mutations. Investigation of the SF3B1K700E interactome identified an enrichment of SF3B1K700E binding with ER, aberrant splicing of ER target genes, global rewiring of ER chromatin binding and resistance to endocrine therapy. Silencing of the aberrantly spliced candidate genes PPIH, TRIM37, HIGD1A, BRD9, and PHKG2 significantly enhanced the growth of the SF3B1 mutant cells, suggestive of a dose dependent tumour suppressive effect. Through synthetic-lethal drug screens we found that SF3B1MUT cells are selectively sensitive to PARP inhibitors. SF3B1MUT cells display a defective response to PARPi induced replication stress. Mechanistically, this occurs via defective ATR signalling in SF3B1MUT cells, which upon PARPi exposure leads to increased replication origin firing and loss of pChk1 (S317) induction. The resultant replication stress leads to failure to resolve DNA replication intermediates via the endonuclease MUS81 and cell cycle stalling at the G2/M checkpoint. These defects can be further targeted by ATM, CDK7 or FACT inhibition, when used in combination with PARPi treatment. This SF3B1MUT selective PARPi sensitivity is preserved across multiple cell lines and patient derived tumour models. In vivo, PARPi produce profound anti-tumour effects in multiple SF3B1MUT cancer models and eliminate distant metastases. Conclusions: Our integrative analysis reveals mechanistic insight into the role of SF3B1 mutations in endocrine therapy response in ER+ breast cancers, where altered SF3B1 induces ER-transcriptional re-programming. We further identified a robust synthetic-lethal relationship of mutant SF3B1 with PARP inhibition that is caused by a defective response to PARPi induced replication stress. Furthermore, we identified several potential selective combination strategies together with PARPi that are selective for SF3B1MUT cells. Together, these data provide the pre-clinical and mechanistic rationale for assessing already-approved PARPi in a biomarker-defined subset of advanced ER+ BC. Table 1. Identified potential therapies for SF3B1 mutant cancers from this study and the literature Citation Format: Phil Bland, Harry Saville, Abigail Read, Patty Wai, Gareth Muirhead, Lucinda Curnow, Jadwiga Nieminuszczy, Nivedita Ravindran, Marie John, Somaieh Hedayat, Holly Barker, James Wright, Lu Yu, Ioanna Mavrommati, Barrie Peck, Mark Allen, Patrycja Gazinska, Helen Pemberton, Aditi Gulati, Sarah Nash, Farzana Noor, Naomi Guppy, Ioannis Roxanis, Samantha Barlow, Helen Kalirai, Sarah Coupland, Ronan Broderick, Samar Alsafadi, Alexandre Houy, Marc-Henri Stern, Stephen Pettit, Jyoti Choudhary, Syed Haider, Wojciech Niedzwiedz, Christopher Lord, Rachael Natrajan. Mutations in the RNA Splicing Factor SF3B1 drive endocrine therapy resistance and confer a targetable replication stress response defect through PARP inhibition. [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-10-05.

Purpose: To identify potential immune targets in post-neoadjuvant chemotherapy (NAC)–resistant triple-negative breast cancer (TNBC) and ER+HER2– breast cancer disease. Experimental Design: Following pathology review, 153 patients were identified as having residual cancer burden (RCB) II/III disease (TNBC n = 80; ER+HER2–n = 73). Baseline pre-NAC samples were available for evaluation for 32 of 80 TNBC and 36 of 73 ER+HER2– cases. Bright-field hematoxylin and eosin assessment allowed for tumor-infiltrating lymphocyte (TIL) evaluation in all cases. Multiplexed immunofluorescence was used to identify the abundance and distribution of immune cell subsets. Levels of checkpoints including PD-1/PD-L1 expression were also quantified. Findings were then validated using expression profiling of cancer and immune-related genes. Cytometry by time-of-flight characterized the dynamic changes in circulating immune cells with NAC. Results: RCB II/III TNBC and ER+HER2– breast cancer were immunologically “cold” at baseline and end of NAC. Although the distribution of immune cell subsets across subtypes was similar, the mRNA expression profiles were both subtype- and chemotherapy-specific. TNBC RCB II/III disease was enriched with genes related to neutrophil degranulation, and displayed strong interplay across immune and cancer pathways. We observed similarities in the dynamic changes in B-cell biology following NAC irrespective of subtype. However, NAC induced changes in the local and circulating tumor immune microenvironment (TIME) that varied by subtype and response. Specifically, in TNBC residual disease, we observed downregulation of stimulatory (CD40/OX40L) and inhibitory (PD-L1/PD-1) receptor expression and an increase in NK cell populations (especially non-cytolytic, exhausted CD56dimCD16–) within both the local TIME and peripheral white cell populations. Conclusions: This study identifies several potential immunologic pathways in residual disease, which may be targeted to benefit high-risk patients.

S3. Expression of mcoEltd1 in 4T1 cells does not affect cell growth in vitro but increases tumour growth and metastasis.

S1. Expression of mcoEltd1 in E0771 cells does not affect cell growth or invasion.

Supplementary Figure and Table Legends

S2. E0771 mcoEltd1 tumours have increased vessel and M2 macrophage markers.

Supplementary Table 1. QPCR primer sequences

S6. Eltd1 increases myeloid accumulation in the spleen.

S4. Eltd1 expressing tumour cells proliferate in the presence of endothelial cells and modifies their secretome.

S5. Representative flow cytometry dot plots indicating the gating strategy for identification of myeloid and lymphoid populations and PD-L1.

ELTD1/ADGRL4 expression is increased in the vasculature of a number of tumor types and this correlates with a good prognosis. Expression has also been reported in some tumor cells with high expression correlating with a good prognosis in hepatocellular carcinoma (HCC) and a poor prognosis in glioblastoma. Here we show that 35% of primary human breast tumors stain positively for ELTD1, with 9% having high expression that correlates with improved relapse-free survival. Using immunocompetent, syngeneic mouse breast cancer models we found that tumors expressing recombinant murine Eltd1 grew faster than controls, with an enhanced ability to metastasize and promote systemic immune effects. The Eltd1-expressing tumors had larger and better perfused vessels and tumor–endothelial cell interaction led to the release of proangiogenic and immune-modulating factors. M2-like macrophages increased in the stroma along with expression of programmed death-ligand 1 (PD-L1) on tumor and immune cells, to create an immunosuppressive microenvironment that allowed Eltd1-regulated tumor growth in the presence of an NY-ESO-1–specific immune response. Eltd1-positive tumors also responded better to chemotherapy which could explain the relationship to a good prognosis observed in primary human cases. Thus, ELTD1 expression may enhance delivery of therapeutic antibodies to reverse the immunosuppression and increase response to chemotherapy and radiotherapy in this subset of tumors. ELTD1 may be useful as a selection marker for such therapies.Implications:ELTD1 expression in mouse breast tumors creates an immunosuppressive microenvironment and increases vessel size and perfusion. Its expression may enhance the delivery of therapies targeting the immune system.

S7. Mice with Eltd1 expressing tumours have less specific T CD8+ cells raised against the tumour NY-ESO-1 antigen.

Supplementary Data from Dynamic Changes in the NK-, Neutrophil-, and B-cell Immunophenotypes Relevant in High Metastatic Risk Post Neoadjuvant Chemotherapy–Resistant Early Breast Cancers

Supplementary Figure from Dynamic Changes in the NK-, Neutrophil-, and B-cell Immunophenotypes Relevant in High Metastatic Risk Post Neoadjuvant Chemotherapy–Resistant Early Breast Cancers

Purpose:To identify potential immune targets in post-neoadjuvant chemotherapy (NAC)–resistant triple-negative breast cancer (TNBC) and ER+HER2– breast cancer disease.Experimental Design:Following pathology review, 153 patients were identified as having residual cancer burden (RCB) II/III disease (TNBC n = 80; ER+HER2–n = 73). Baseline pre-NAC samples were available for evaluation for 32 of 80 TNBC and 36 of 73 ER+HER2– cases. Bright-field hematoxylin and eosin assessment allowed for tumor-infiltrating lymphocyte (TIL) evaluation in all cases. Multiplexed immunofluorescence was used to identify the abundance and distribution of immune cell subsets. Levels of checkpoints including PD-1/PD-L1 expression were also quantified. Findings were then validated using expression profiling of cancer and immune-related genes. Cytometry by time-of-flight characterized the dynamic changes in circulating immune cells with NAC.Results:RCB II/III TNBC and ER+HER2– breast cancer were immunologically “cold” at baseline and end of NAC. Although the distribution of immune cell subsets across subtypes was similar, the mRNA expression profiles were both subtype- and chemotherapy-specific. TNBC RCB II/III disease was enriched with genes related to neutrophil degranulation, and displayed strong interplay across immune and cancer pathways. We observed similarities in the dynamic changes in B-cell biology following NAC irrespective of subtype. However, NAC induced changes in the local and circulating tumor immune microenvironment (TIME) that varied by subtype and response. Specifically, in TNBC residual disease, we observed downregulation of stimulatory (CD40/OX40L) and inhibitory (PD-L1/PD-1) receptor expression and an increase in NK cell populations (especially non-cytolytic, exhausted CD56dimCD16–) within both the local TIME and peripheral white cell populations.Conclusions:This study identifies several potential immunologic pathways in residual disease, which may be targeted to benefit high-risk patients.

Enrichment analysis from TCGA RNA-seq and RPPA data comparing CREBBPaltered versus wild-type patients.

Supplementary Figures and legends

Triple-negative breast cancers (TNBC) are resistant to standard-of-care chemotherapy and lack known targetable driver gene alterations. Identification of novel drivers could aid the discovery of new treatment strategies for this hard-to-treat patient population, yet studies using high-throughput and accurate models to define the functions of driver genes in TNBC to date have been limited. Here, we employed unbiased functional genomics screening of the 200 most frequently mutated genes in breast cancer, using spheroid cultures to model in vivo–like conditions, and identified the histone acetyltransferase CREBBP as a novel tumor suppressor in TNBC. CREBBP protein expression in patient tumor samples was absent in 8% of TNBCs and at a high frequency in other tumors, including squamous lung cancer, where CREBBP-inactivating mutations are common. In TNBC, CREBBP alterations were associated with higher genomic heterogeneity and poorer patient survival and resulted in upregulation and dependency on a FOXM1 proliferative program. Targeting FOXM1-driven proliferation indirectly with clinical CDK4/6 inhibitors (CDK4/6i) selectively impaired growth in spheroids, cell line xenografts, and patient-derived models from multiple tumor types with CREBBP mutations or loss of protein expression. In conclusion, we have identified CREBBP as a novel driver in aggressive TNBC and identified an associated genetic vulnerability in tumor cells with alterations in CREBBP and provide a preclinical rationale for assessing CREBBP alterations as a biomarker of CDK4/6i response in a new patient population.Significance:This study demonstrates that CREBBP genomic alterations drive aggressive TNBC, lung cancer, and lymphomas and may be selectively treated with clinical CDK4/6 inhibitors.

Supplementary Materials and Methods

With a median survival of 4 months, the development of breast cancer leptomeningeal metastasis (BCLM) is devastating for patients. Investigating this often occult disease process is hampered by the difficulty of accessing leptomeningeal tumour material. Here, we utilise cerebrospinal fluid as a source of both cell-free DNA (cfDNA) and disseminated tumour cells, to explore the evolution of BCLM and heterogeneity between leptomeningeal and extracranial sites. CSF cfDNA sequencing detected actionable somatic alterations in 81% (17/21) of BCLM cases. Further genomic characterisation identified frequently altered genes involved in cell adherens junctions and cytoskeleton/chemotaxis pathways. Patient-derived organoids established from CSF tumour cells allowed profiling of this rarely available biological material, in addition to therapeutic testing and generation ofin vivomodels. Together these data reveal the development of BCLM is associated with the acquisition of a lobular-like breast cancer phenotype, and highlight potential approaches for tackling this hard-to-treat form of metastatic disease.

Necroptosis is a lytic, inflammatory form of cell death that not only contributes to pathogen clearance but can also lead to disease pathogenesis. Necroptosis is triggered by RIPK3-mediated phosphorylation of MLKL, which is thought to initiate MLKL oligomerisation, membrane translocation and membrane rupture, although the precise mechanism is incompletely understood. Here, we show that K63-linked ubiquitin chains are attached to MLKL during necroptosis and that ubiquitylation of MLKL at K219 significantly contributes to the cytotoxic potential of phosphorylated MLKL. The K219R MLKL mutation protects animals from necroptosis-induced skin damage and renders cells resistant to pathogen-induced necroptosis. Mechanistically, we show that ubiquitylation of MLKL at K219 is required for higher-order assembly of MLKL at membranes, facilitating its rupture and necroptosis. We demonstrate that K219 ubiquitylation licenses MLKL activity to induce lytic cell death, suggesting that necroptotic clearance of pathogens as well as MLKL-dependent pathologies are influenced by the ubiquitin-signalling system., Necroptosis is a form of cell death characterized by membrane rupture via MLKL oligomerization, although mechanistic details remain unclear. Here, the authors show that MLKL ubiquitylation of K219 facilitates high-order membrane assembly and subsequent rupture, promoting cytotoxicity.

Background: Assessing how clinical resistance to PARP inhibitors develops has been challenging, due to the lack of coverage of potential resistance genes in sequencing panels and biopsies being subject to spatial heterogeneity. We studied the development of PARPi and/or platinum resistant disease using both tissue and a novel liquid biopsy assay, in patients treated for BRCA1/2-mutated metastatic breast cancer (BRCA1/2m mBC). Approach: A cohort of 35 mBC patients with germline or somatic BRCA1/2 mutations were identified as having developed PARPi or platinum resistant disease. Tumour biopsies were analysed by exome and transcriptome sequencing whereas ctDNA isolated from plasma sampled across the pre-treatment, response and eventual progression journey were analysed using the Guardant INFINITY platform equipped to detect mutations in >800 genes, and genome wide methylation, including promoter methylation in 398 cancer-related genes. Somatic mutations or regions of methylation that were associated with resistance were identified, including BRCA1/2 reversion mutations. Somatic mutations with the potential to cause PARPi resistance were annotated using CRISPR screen data and other functional analyses describing genes that alter PARPi synthetic lethality. Results: The most common resistance mechanism was BRCA1/2 reversion mutation (51%; n=8 BRCA1m patients, n=10 BRCA2m patients). Most reversions (77%) occurred via deletions (BRCA1, 70%; BRCA2, 79%) and exhibited microhomology use at junctions (> 80% for both BRCA1 and BRCA2). In 14 patients, multiple concurrent reversion mutations were detected with VAFs ranging from 0.1-40% for BRCA1 and 0.05-18% for BRCA2. Changes in VAF over time indicated that different reversions in the same patient may impart different fitness advantages in the face of treatment. Loss-of-function mutations in the 53BP1-Shieldin pathway were identified in two BRCA1m patients, and VUS mutations in the pathway were seen in two further patients. In some patients, reversion mutations co-occurred with either 53BP1-Shieldin pathway mutations or with replication fork stability mutations (PAXIP1 in BRCA2m patients) - indicating concurrent but mechanistically distinct forms of resistance develop in the same patient. We isolated a PDX model from one patient that exhibits resistance via both BRCA1 reversion and loss of 53BP1, allowing this phenomenon to be modelled in more detail. We also detected BRCA1 methylation in ctDNA in a patient with a somatic rearrangement in BRCA1. Conclusions: Liquid biopsy profiling of PARPi-resistance breast cancer patients indicates that many patients develop multiple reversion mutations, and that alterations in the 53BP1-Shieldin pathway are present but less frequent. The co-occurrence of 53BP1-Shieldin pathway mutations and reversion mutations suggests parallel mechanisms of resistance can operate in the same patient. Citation Format: Elizabeth Harvey-Jones, Maya Raghunandan, Luisa Robbez-Masson, Alaguthurai Thanussuyah, Roberta Liccardo, Arielle Yablonovitch, Mingyang Cai, Leylah Drusbosky, Michael Dorschner, Lorena Magraner Pardo, Rebecca Marlow, Asha Konde, Jennifer Trendell, John Alexander, Syed Haider, Chris Starling, Ioannis Roxanis, Jennifer Yen, Stephen J. Pettitt, Christopher J. Lord, Andrew N. Tutt. Longitudinal analysis of PARP inhibitor and platinum resistance in BRCA1/2m breast cancer using liquid biopsy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6094.

Background The TNT trial (NCT00532727) showed no evidence of carboplatin (C) superiority over docetaxel (D) overall in metastatic triple negative breast cancers (TNBC), but a C benefit was observed in the pre-specified sub-group analysis in patients with a gBRCA1/2 mutation (Tutt et al, Nat Med 2018). Given only ~30% of patients have a gBRCA1/2 mutation, broader predictive biomarkers of response are needed. In this cohort we previously found that DNA Damage Response (DDR) signatures were associated with improved C response in chemotherapy (CT) naïve patients only (Tovey et al, ASCO 2020). Since DDR activities influence tumour immune-microenvironment, we explored the predictive ability of immune cell markers and performed integrative analyses on multi-omics features to identify novel TNBC subgroups. Patients and Methods Tumour infiltrating lymphocytes (TILs) were evaluated on haematoxylin and eosin stained primary tumour (PT) slides for 222/376 TNT patients. Formalin-fixed paraffin-embedded PT tissues from 186/376 TNT patients were successfully profiled using total RNA-sequencing. Matched recurrence (REC) was also sequenced for 13 patients. Twenty-five immune signatures were assessed. Logistic regression and restricted mean progression free survival (PFS) were applied to delineate the relationship of these features with treatment outcomes. Random forest clustering of multi-omics DDR and immune biology markers, including gene expression signatures and mutation/methylation status, was applied to identify subgroups. We further molecularly characterised these clusters through supervised clustering of 693 gene expression “modules” (sets of co-expressed genes), immune cell deconvolution and genomic scars. Results Immune gene expression signatures and TILs were highly correlated. Average immune infiltration based on ConsensusTME was lower in mutated/methylated tumours compared with BRCA1 wildtype tumours (p=0.04). Immune signature score markers decreased from PT to REC, demonstrating a dynamic immune microenvironment. In the overall population and when restricting to prior CT treated patients, high immune infiltration (gene expression based & TILs) was associated with response to D while C response rates were not associated with immune scores (interaction p-values< 0.05). This did not translate to a PFS benefit. Multi-omics clustering identified 6 biological subgroups including immune enriched, immune depleted, DDR deficient and proficient clusters as well as 2 small clusters with no obvious distinguishing features. Immune enriched TNBC were predominantly basal-like immune activated with high B-cell/T-cell diversity. Immune depleted TNBC showed higher activity of proliferation and DDR pathway modules. DDR proficient tumours had low expression of immune markers and enrichment for ESR1/PGR expression, markers of extra cellular formation, cell structure, lipid metabolism and proliferation. The DDR deficient cluster was enriched for proliferation and demonstrated high number of TILs despite no apparent enrichment for gene expression-based immune modules. In the prior CT treated cohort, the immune enriched cluster had preferential response to D (62.5% (D) vs. 29.4% (C); p=0.02). The immune depleted cluster had preferential response to C (8.0% (D) vs. 40.0% (C); p=0.01). Numbers were too small to assess differential response within the other clusters or in the CT naïve cohort. Conclusions Tumours with high immune features have high response to D while those with low immune features have preferential response to C in advanced TNBC. Combining multi-omics markers of DDR deficiency and immune biology can identify clusters of patients with distinct biological profiles and differential treatment specific response rates. Citation Format: Holly Tovey, Orsolya Sipos, Katherine A Hoadley, Joel S Parker, Jelmar Quist, Sarah Kernaghan, Lucy Kilburn, Roberto Salgado, Sherene Loi, Richard D Kennedy, Ioannis Roxanis, Patrycja Gazinska, Sarah E. Pinder, Judith Bliss, Charles M. Perou, Syed Haider, Andrew Tutt, Anita Grigoriadis, Maggie Chon U Cheang. Histopathological and molecular immune landscape and DNA damage response signatures to predict response to carboplatin and docetaxel in TNT trial TNBC cohort [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD9-06.

Simple Summary Invasive lobular breast cancers (ILCs) are histologically classified by their discohesive growth pattern, due to loss of the cell adhesion glycoprotein E-cadherin (CDH1), which arises via mutation in CDH1 in around half of these tumours. A subset of these tumours, however, show mixed levels of E-cadherin expression. Here, we sought to address whether the distinct parts of individual tumours showing heterogeneous E-cadherin expression harbour distinct driver alterations. Using whole genome sequencing and methylation profiling of nine such cases, we identified that these tumours are clonally related, suggesting that they are part of the spectrum of ILC tumours. CDH1 mutant tumours showed a higher mutational burden indicative of APOBEC-mediated mutagenesis. In some cases, known clinically actionable driver mutations, such as PIK3CA, were exclusive to one component. Together, these results highlight the heterogeneity underpinning this special histological breast cancer. Abstract 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.

Our breast screening unit was identified as high outlier for B3 lesions with a low positive predictive value (PPV) compared to the England average. This prompted a detailed internal audit and review of B3 lesions and their outcomes to identify causes and address any variation in practice.The B3 rate was calculated in 4168 breast core biopsies from 2019, using the subsequent excision to determine the PPV. Atypical intraductal epithelial proliferation (AIDEP) cases were subject to microscopic review to reassess the presence of atypia against published criteria. The B3 rate was re-audited in 2021, and the results compared.Screening cases had a high B3 rate of 12.4% (30% above the national average), and a PPV of 7.7% (9.7% with atypia). AIDEP was identified as a possible cause of this outlier status. On review and by consensus, AIDEP was confirmed in only 66% of cases reported as such, 17% were downgraded, and 16% did not reach consensus, the latter highlighting the difficulty and subjectivity in diagnosis of these lesions. Repeat audit of B3 rates after this extended review revealed a reduction from 12.4% to 9.11%, which is more in line with national standards.Benchmarking against national reporting standards is critical for service improvement. Through a supportive environment, team working, rigorous internal review and adherence to guidelines, interobserver variation and outlier status in breast pathology screening outliers can both be addressed. This study can serve as a model to other outlier units to identify and tackle underlying causes.

ELTD1/ADGRL4 expression is increased in the vasculature of a number of tumor types and this correlates with a good prognosis. Expression has also been reported in some tumor cells with high expression correlating with a good prognosis in hepatocellular carcinoma (HCC) and a poor prognosis in glioblastoma. Here we show that 35% of primary human breast tumors stain positively for ELTD1, with 9% having high expression that correlates with improved relapse-free survival. Using immunocompetent, syngeneic mouse breast cancer models we found that tumors expressing recombinant murine Eltd1 grew faster than controls, with an enhanced ability to metastasize and promote systemic immune effects. The Eltd1-expressing tumors had larger and better perfused vessels and tumor–endothelial cell interaction led to the release of proangiogenic and immune-modulating factors. M2-like macrophages increased in the stroma along with expression of programmed death-ligand 1 (PD-L1) on tumor and immune cells, to create an immunosuppressive microenvironment that allowed Eltd1-regulated tumor growth in the presence of an NY-ESO-1–specific immune response. Eltd1-positive tumors also responded better to chemotherapy which could explain the relationship to a good prognosis observed in primary human cases. Thus, ELTD1 expression may enhance delivery of therapeutic antibodies to reverse the immunosuppression and increase response to chemotherapy and radiotherapy in this subset of tumors. ELTD1 may be useful as a selection marker for such therapies. Implications: ELTD1 expression in mouse breast tumors creates an immunosuppressive microenvironment and increases vessel size and perfusion. Its expression may enhance the delivery of therapies targeting the immune system.

Triple-negative breast cancers (TNBC) are resistant to standard-of-care chemotherapy and lack known targetable driver gene alterations. Identification of novel drivers could aid the discovery of new treatment strategies for this hard-to-treat patient population, yet studies using high-throughput and accurate models to define the functions of driver genes in TNBC to date have been limited. Here, we employed unbiased functional genomics screening of the 200 most frequently mutated genes in breast cancer, using spheroid cultures to model in vivo–like conditions, and identified the histone acetyltransferase CREBBP as a novel tumor suppressor in TNBC. CREBBP protein expression in patient tumor samples was absent in 8% of TNBCs and at a high frequency in other tumors, including squamous lung cancer, where CREBBP-inactivating mutations are common. In TNBC, CREBBP alterations were associated with higher genomic heterogeneity and poorer patient survival and resulted in upregulation and dependency on a FOXM1 proliferative program. Targeting FOXM1-driven proliferation indirectly with clinical CDK4/6 inhibitors (CDK4/6i) selectively impaired growth in spheroids, cell line xenografts, and patient-derived models from multiple tumor types with CREBBP mutations or loss of protein expression. In conclusion, we have identified CREBBP as a novel driver in aggressive TNBC and identified an associated genetic vulnerability in tumor cells with alterations in CREBBP and provide a preclinical rationale for assessing CREBBP alterations as a biomarker of CDK4/6i response in a new patient population. Significance: This study demonstrates that CREBBP genomic alterations drive aggressive TNBC, lung cancer, and lymphomas and may be selectively treated with clinical CDK4/6 inhibitors.

This work provides a quantitative tool for the analysis of fibrillar patterns in biology. We demonstrates its utility on in vitro generated extracellular matrices, mouse tissues, and patient samples. The tool is already publicly available to the research community., Diverse extracellular matrix patterns are observed in both normal and pathological tissue. However, most current tools for quantitative analysis focus on a single aspect of matrix patterning. Thus, an automated pipeline that simultaneously quantifies a broad range of metrics and enables a comprehensive description of varied matrix patterns is needed. To this end, we have developed an ImageJ plugin called TWOMBLI, which stands for The Workflow Of Matrix BioLogy Informatics. This pipeline includes metrics of matrix alignment, length, branching, end points, gaps, fractal dimension, curvature, and the distribution of fibre thickness. TWOMBLI is designed to be quick, versatile and easy-to-use particularly for non-computational scientists. TWOMBLI can be downloaded from https://github.com/wershofe/TWOMBLI together with detailed documentation and tutorial video. Although developed with the extracellular matrix in mind, TWOMBLI is versatile and can be applied to vascular and cytoskeletal networks. Here we present an overview of the pipeline together with examples from a wide range of contexts where matrix patterns are generated.

High computational cost associated with digital pathology image analysis approaches is a challenge towards their translation in routine pathology clinic. Here, we propose a computationally efficient framework (SuperHistopath), designed to map global context features reflecting the rich tumor morphological heterogeneity. SuperHistopath efficiently combines i) a segmentation approach using the linear iterative clustering (SLIC) superpixels algorithm applied directly on the whole-slide images at low resolution (5x magnification) to adhere to region boundaries and form homogeneous spatial units at tissue-level, followed by ii) classification of superpixels using a convolution neural network (CNN). To demonstrate how versatile SuperHistopath was in accomplishing histopathology tasks, we classified tumor tissue, stroma, necrosis, lymphocytes clusters, differentiating regions, fat, hemorrhage and normal tissue, in 127 melanomas, 23 triple-negative breast cancers, and 73 samples from transgenic mouse models of high-risk childhood neuroblastoma with high accuracy (98.8%, 93.1% and 98.3% respectively). Furthermore, SuperHistopath enabled discovery of significant differences in tumor phenotype of neuroblastoma mouse models emulating genomic variants of high-risk disease, and stratification of melanoma patients (high ratio of lymphocyte-to-tumor superpixels (p = 0.015) and low stroma-to-tumor ratio (p = 0.028) were associated with a favorable prognosis). Finally, SuperHistopath is efficient for annotation of ground-truth datasets (as there is no need of boundary delineation), training and application (~5 min for classifying a whole-slide image and as low as ~30 min for network training). These attributes make SuperHistopath particularly attractive for research in rich datasets and could also facilitate its adoption in the clinic to accelerate pathologist workflow with the quantification of phenotypes, predictive/prognosis markers.

Diverse extracellular matrix patterns are observed in both normal and pathological tissue. However, most current tools for quantitative analysis focus on a single aspect of matrix patterning. Thus, an automated pipeline that simultaneously quantifies a broad range of metrics and enables a comprehensive description of varied matrix patterns is needed. To this end we have developed an ImageJ plugin called TWOMBLI, which stands for The Workflow Of Matrix BioLogy Informatics. TWOMBLI is designed to be quick, versatile and easy-to-use particularly for non-computational scientists. TWOMBLI can be downloaded from https://github.com/wershofe/TWOMBLI together with detailed documentation. Here we present an overview of the pipeline together with examples from a wide range of contexts where matrix patterns are generated.

Simple Summary Ovarian clear cell carcinoma (OCCC) is a rare subtype of epithelial ovarian cancer that has a poor response to chemotherapy. Here, we assessed the immunological features of a series of 33 OCCCs and identified an immune-related gene expression signature that correlated with a patient’s risk of recurrence. Additionally, using multiplex immunofluorescence, we assessed the spatial distribution and abundance of immune cell populations at the protein level and identified that tumour-associated macrophages (TAM) and regulatory T cells are excluded from the vicinity of tumour cells in low-risk patients, suggesting that high-risk patients have a more immunosuppressive microenvironment. We also found that TAMs and cytotoxic T cells were also excluded from the vicinity of tumour cells in ARID1A mutated OCCCs, suggesting that the exclusion of these immune effectors could determine the host response in ARID1A mutant OCCCs. Abstract Ovarian clear cell carcinoma (OCCC) is a rare subtype of epithelial ovarian cancer characterised by a high frequency of loss-of-function ARID1A mutations and a poor response to chemotherapy. Despite their generally low mutational burden, an intratumoural T cell response has been reported in a subset of OCCC, with ARID1A purported to be a biomarker for the response to the immune checkpoint blockade independent of micro-satellite instability (MSI). However, assessment of the different immune cell types and spatial distribution specifically within OCCC patients has not been described to date. Here, we characterised the immune landscape of OCCC by profiling a cohort of 33 microsatellite stable OCCCs at the genomic, gene expression and histological level using targeted sequencing, gene expression profiling using the NanoString targeted immune panel, and multiplex immunofluorescence to assess the spatial distribution and abundance of immune cell populations at the protein level. Analysis of these tumours and subsequent independent validation identified an immune-related gene expression signature associated with risk of recurrence of OCCC. Whilst histological quantification of tumour-infiltrating lymphocytes (TIL, Salgado scoring) showed no association with the risk of recurrence or ARID1A mutational status, the characterisation of TILs via multiplexed immunofluorescence identified spatial differences in immunosuppressive cell populations in OCCC. Tumour-associated macrophages (TAM) and regulatory T cells were excluded from the vicinity of tumour cells in low-risk patients, suggesting that high-risk patients have a more immunosuppressive microenvironment. We also found that TAMs and cytotoxic T cells were also excluded from the vicinity of tumour cells in ARID1A-mutated OCCCs compared to ARID1A wild-type tumours, suggesting that the exclusion of these immune effectors could determine the host response of ARID1A-mutant OCCCs to therapy. Overall, our study has provided new insights into the immune landscape and prognostic associations in OCCC and suggest that tailored immunotherapeutic approaches may be warranted for different subgroups of OCCC patients.

Drugs that mobilise the immune system against cancer are dramatically improving care for many people. Dying cancer cells play an active role in inducing anti‐tumour immunity but not every form of death can elicit an immune response. Moreover, resistance to apoptosis is a major problem in cancer treatment and disease control. While the term “immunogenic cell death” is not fully defined, activation of receptor‐interacting serine/threonine‐protein kinase 1 (RIPK1) can induce a type of death that mobilises the immune system against cancer. However, no clinical treatment protocols have yet been established that would harness the immunogenic potential of RIPK1. Here, we report the first pre‐clinical application of an in vivo treatment protocol for soft‐tissue sarcoma that directly engages RIPK1‐mediated immunogenic cell death. We find that RIPK1‐mediated cell death significantly improves local disease control, increases activation of CD8+ T cells as well as NK cells, and enhances the survival benefit of immune checkpoint blockade. Our findings warrant a clinical trial to assess the survival benefit of RIPK1‐induced cell death in patients with advanced disease at limb extremities., The pathways that initiate and execute immunogenic cell death are complex, genetically encoded, and subject to significant regulation. This study focusses on the possibility to engage RIPK1‐mediated immunogenic cell death for the treatment of soft tissue sarcomas that occur at limb extremities.

Collagen plays a key role in normal and malignant tissue homeostasis. While the prognostic significance of collagen fiber remodeling in invasive breast cancer has been studied, its role in ductal carcinoma in situ (DCIS) remains poorly defined. Using image analysis, we aimed to evaluate the prognostic significance of the geometric characteristics of collagen surrounding DCIS. A large well-characterized cohort of DCIS comprising pure DCIS (n = 610) and DCIS coexisting with invasive carcinoma (n = 180) were histochemically stained for collagen using picrosirius red. ImageJ software was used to assess collagen density, degree of collagen fiber dispersion and directionality in relation to DCIS ducts’ boundary. We developed a collagen prognostic index and evaluated its prognostic significance. A poor index was observed in 24% of the pure DCIS and was associated with determinants of high-risk DCIS including higher nuclear grade, comedo type necrosis, hormonal receptor negativity, HER2 positivity and high proliferation index. High collagen prognostic index was associated with the collagen remodeling protein prolyl-4-hydroxlase alpha subunit 2 and the hypoxia-related protein hypoxia inducible factor 1α. DCIS coexisting with invasive breast cancer had a higher collagen prognostic index than pure DCIS ( p

Late-phase clinical trials investigating metformin as a cancer therapy are underway. However, there remains controversy as to the mode of action of metformin in tumors at clinical doses. We conducted a clinical study integrating measurement of markers of systemic metabolism, dynamic FDG-PET-CT, transcriptomics, and metabolomics at paired time points to profile the bioactivity of metformin in primary breast cancer. We show metformin reduces the levels of mitochondrial metabolites, activates multiple mitochondrial metabolic pathways, and increases 18-FDG flux in tumors. Two tumor groups are identified with distinct metabolic responses, an OXPHOS transcriptional response (OTR) group for which there is an increase in OXPHOS gene transcription and an FDG response group with increased 18-FDG uptake. Increase in proliferation, as measured by a validated proliferation signature, suggested that patients in the OTR group were resistant to metformin treatment. We conclude that mitochondrial response to metformin in primary breast cancer may define anti-tumor effect.

The field of immuno-oncology has expanded rapidly over the past decade, but key questions remain. How does tumour-immune interaction regulate disease progression? How can we prospectively identify patients who will benefit from immunotherapy? Identifying measurable features of the tumour immune-microenvironment which have prognostic or predictive value will be key to making meaningful gains in these areas. Recent developments in deep learning enable big-data analysis of pathological samples. Digital approaches allow data to be acquired, integrated and analysed far beyond what is possible with conventional techniques, and to do so efficiently and at scale. This has the potential to reshape what can be achieved in terms of volume, precision and reliability of output, enabling data for large cohorts to be summarised and compared. This review examines applications of artificial intelligence (AI) to important questions in immuno-oncology (IO). We discuss general considerations that need to be taken into account before AI can be applied in any clinical setting. We describe AI methods that have been applied to the field of IO to date and present several examples of their use.

Cancer-associated fibroblasts (CAFs) play a key role in promoting tumor growth, acting through complex paracrine regulation. GTP cyclohydrolase (GTPCH) expression for tetrahydrobiopterin synthesis in tumor stroma is implicated in angiogenesis and tumor development. However, the clinical significance of GTPCH expression in breast cancer is still elusive and how GTPCH regulates stromal fibroblast and tumor cell communication remains unknown. We found that GTPCH was upregulated in breast CAFs and epithelia, and high GTPCH RNA was significantly correlated with larger high grade tumors and worse prognosis. In cocultures, GTPCH expressing fibroblasts stimulated breast cancer cell proliferation and motility, cancer cell Tie2 phosphorylation and consequent downstream pathway activation. GTPCH interacted with Ang-1 in stromal fibroblasts and enhanced Ang-1 expression and function, which in turn phosphorylated tumor Tie2 and induced cell proliferation. In coimplantation xenografts, GTPCH in fibroblasts enhanced tumor growth, upregulating Ang-1 and alpha-smooth muscle actin mainly in fibroblast-like cells. GTPCH inhibition resulted in the attenuation of tumor growth and angiogenesis. GTPCH/Ang-1 interaction in stromal fibroblasts and activation of Tie2 on breast tumor cells could play an important role in supporting breast cancer growth. GTPCH may be an important mechanism of paracrine tumor growth and hence a target for therapy in breast cancer.

Fatty acid binding protein 4 (FABP4) is a fatty acid chaperone, which is induced during adipocyte differentiation. Previously we have shown that FABP4 in endothelial cells is induced by the NOTCH1 signalling pathway, the latter of which is involved in mechanisms of resistance to antiangiogenic tumour therapy. Here, we investigated the role of FABP4 in endothelial fatty acid metabolism and tumour angiogenesis. We analysed the effect of transient FABP4 knockdown in human umbilical vein endothelial cells on fatty acid metabolism, viability and angiogenesis. Through therapeutic delivery of siRNA targeting mouse FABP4, we investigated the effect of endothelial FABP4 knockdown on tumour growth and blood vessel formation. In vitro, siRNA-mediated FABP4 knockdown in endothelial cells led to a marked increase of endothelial fatty acid oxidation, an increase of reactive oxygen species and decreased angiogenesis. In vivo, we found that increased NOTCH1 signalling in tumour xenografts led to increased expression of endothelial FABP4 that decreased when NOTCH1 and VEGFA inhibitors were used in combination. Angiogenesis, growth and metastasis in ovarian tumour xenografts were markedly inhibited by therapeutic siRNA delivery targeting mouse endothelial FABP4. Therapeutic targeting of endothelial FABP4 by siRNA in vivo has antiangiogenic and antitumour effects with minimal toxicity and should be investigated further.

The spectacular response observed in clinical trials of immunotherapy in patients with previously uncurable Melanoma, a highly aggressive form of skin cancer, calls for a better understanding of the cancer-immune interface. Computational pathology provides a unique opportunity to spatially dissect such interface on digitised pathological slides. Accurate cellular classification is a key to ensure meaningful results, but is often challenging even with state-of-art machine learning and deep learning methods.

Angiosarcoma is an aggressive malignancy that arises spontaneously or secondarily to ionizing radiation or chronic lymphoedema. Previous work has identified aberrant angiogenesis, including occasional somatic mutations in angiogenesis signaling genes, as a key driver of angiosarcoma. Here we employed whole-genome, whole-exome and targeted sequencing to study the somatic changes underpinning primary and secondary angiosarcoma. We identified recurrent mutations in two genes, PTPRB and PLCG1, which are intimately linked to angiogenesis. The endothelial phosphatase PTPRB, a negative regulator of vascular growth factor tyrosine kinases, harbored predominantly truncating mutations in 10 of 39 tumors (26%). PLCG1, a signal transducer of tyrosine kinases, encoded a recurrent, likely activating p.Arg707Gln missense variant in 3 of 34 cases (9%). Overall, 15 of 39 tumors (38%) harbored at least one driver mutation in angiogenesis signaling genes. Our findings inform and reinforce current therapeutic efforts to target angiogenesis signaling in angiosarcoma.

Trastuzumab has been shown to improve the survival outcomes of HER2 positive breast cancer patients. However, a significant proportion of HER2-positive patients are either inherently resistant or develop resistance to trastuzumab. We assessed the effects of neratinib, an irreversible panHER inhibitor, in a panel of 36 breast cancer cell lines. We further assessed its effects with or without trastuzumab in several sensitive and resistant breast cancer cells as well as a BT474 xenograft model. We confirmed that neratinib was significantly more active in HER2-amplified than HER2 non-amplified cell lines. Neratinib decreased the activation of the 4 HER receptors and inhibited downstream pathways. However, HER3 and Akt were reactivated at 24 hours, which was prevented by the combination of trastuzumab and neratinib. Neratinib also decreased pHER2 and pHER3 in acquired trastuzumab resistant cells. Neratinib in combination with trastuzumab had a greater growth inhibitory effect than either drug alone in 4 HER2 positive cell lines. Furthermore, trastuzumab in combination with neratinib was growth inhibitory in SKBR3 and BT474 cells which had acquired resistance to trastuzumab as well as in a BT474 xenograft model. Innately trastuzumab resistant cell lines showed sensitivity to neratinib, but the combination did not enhance response compared to neratinib alone. Levels of HER2 and phospho-HER2 showed a direct correlation with sensitivity to neratinib. Our data indicate that neratinib is an effective anti-HER2 therapy and counteracted both innate and acquired trastuzumab resistance in HER2 positive breast cancer. Our results suggest that combined treatment with trastuzumab and neratinib is likely to be more effective than either treatment alone for both trastuzumab-sensitive breast cancer as well as HER2-positive tumors with acquired resistance to trastuzumab.

Metaplastic breast carcinomas are histologically heterogeneous and rare tumours constituting In situ evidence implicating the myoepithelial cell as cell of origin is presented and discussed in view of recent in vitro studies using immortalized human mammary stem/progenitor cell lines.

By contrast with developmental epithelial-mesenchymal transition (EMT), where epithelial characteristics undergo transformation to a mesenchymal-like phenotype in a coordinated fashion, oncogenic EMT occurs in the context of unpredictable genetic changes present in the tumour cells, as well as an abnormal tumour microenvironment. Therefore, a partial form of EMT has been proposed as variably participating in the establishment of invasive phenotype in different types of breast carcinoma, in keeping with their morphological and phenotypical diversity. A complex network of signalling pathways and transcription factors appears responding to various growth factors and cytokines released by stromal and neoplastic elements, endowing the system with abundant regulatory opportunities. The process of EMT is largely elusive in histopathological preparations, prompting doubts regarding its significance in tumour progression. This might be related to the presumed focal occurrence of EMT in the majority of tumours. Detailed topological studies might facilitate understanding of the orchestration of events taking place in vivo. Even more importantly, clinical correlations can be endeavoured and, in parallel with advancement in molecular pathology, a contribution to taxonomy refinement can be envisaged.