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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.

The naked mole rat (NMR), Heterocephalus glaber, the longest-living rodent1, provides a unique opportunity to explore how evolution has shaped adult stem cell (ASC) activity with increasing lifespan. Using cumulative BrdU labelling and a quantitative imaging approach to track intestinal ASCs (Lgr5+) in NMRs over time in their native in vivo state, we show adaptations to living in near-anaerobic2, food-deprived3 subterranean environment, has necessitated an expanded pool of Lgr5+ cells, which exhibit slower division rates compared to short-lived mice, but have similar turnover as humans. Unlike human LGR5+ cells at the crypt base (LGR5+CBC) that slow down their division rates by entering quiescence(G0) 4, NMR Lgr5+CBC cells in both the small intestine and the colon transit slowly through the active cell cycle (G1 to M) by prolonging arrest at G1 and/or G2. The inverse correlation between ASC division rates and lifespan appears to be specific to Lgr5+CBC/LGR5+CBC cells as this relationship is not seen for cells outside the crypt base. Our data provide an explanation for the observed anticorrelation between somatic mutation rates and longevity in mammals5, with ASC lineages in longer-lived organisms turning over slowly to possibly mitigate ASC exhaustion and reduce mutation rates for long-term tissue maintenance. Finally, we also observe an expanded pool of differentiated cells in NMRs that confer enhanced protection and function to the intestinal mucosa which is able to detect any chemical imbalance in the luminal environment efficiently, triggering a robust anti-proliferative, pro-apoptotic response within the stem cell zone.

Lagging chromosomes in E-cadherin defective cell exposed to foretinib leading to failed cytokinesis

Supplementary Figure S1. Frequency of genomic aberrations in the KCL TNBC cohort. Supplementary Figure S2. Conceptual diagrams of Scores of Chromosomal Instability Scarring (SCINS). Supplementary Figure S3. Extent and location of SCINS in TNBC. Supplementary Figure S4. Chromosome-wise distribution of SCINS across four TNBC cohorts. Supplementary Figure S5. SCINS-defined tumour classes identify HORMAD1 to be among the top genes linked with Hi-AiCNA/Hi-CnLOH in PrECOG TNBCs. Supplementary Figure S6. Gene expression signatures and HORMAD1 expression. Supplementary Figure S7. Immunofluorescent localisation of HORMAD1 in cell lines. Supplementary Figure S8. Expression of HORMAD1 in breast cancer cell lines and tumour samples. Supplementary Figure S9. HORMAD1 inhibits activity of the DR-GFP HR reporter. Supplementary Figure S10. Effect of HORMAD1 overexpression and knockdown on cell growth and proliferation. Supplementary Figure S11. HORMAD1 promotes activity of the EJ5 NHEJ reporter. Supplementary Figure S12. Effect of HORMAD1 knockdown on 53BP1 and RAD51 foci in HCC1143. Supplementary Figure S13. BRCA1/2 mutation, platinum sensitivity and genomic scarring or HORMAD1 expression. Supplementary Figure S14. Effect of HORMAD1 knockdown on drug sensitivity in HCC1143. Supplementary Figure S15. ROC analysis of PrECOG TNBCs. Supplementary Figure S16. HORMAD1 expression in unselected and BRCA1/2 wildtype TNBCs.

Supplementary Methods, Figure Legends, Table Legends

The cell adhesion glycoprotein E-cadherin (CDH1) is commonly inactivated in breast tumors. Precision medicine approaches that exploit this characteristic are not available. Using perturbation screens in breast tumor cells with CRISPR/Cas9-engineered CDH1 mutations, we identified synthetic lethality between E-cadherin deficiency and inhibition of the tyrosine kinase ROS1. Data from large-scale genetic screens in molecularly diverse breast tumor cell lines established that the E-cadherin/ROS1 synthetic lethality was not only robust in the face of considerable molecular heterogeneity but was also elicited with clinical ROS1 inhibitors, including foretinib and crizotinib. ROS1 inhibitors induced mitotic abnormalities and multinucleation in E-cadherin–defective cells, phenotypes associated with a defect in cytokinesis and aberrant p120 catenin phosphorylation and localization. In vivo, ROS1 inhibitors produced profound antitumor effects in multiple models of E-cadherin–defective breast cancer. These data therefore provide the preclinical rationale for assessing ROS1 inhibitors, such as the licensed drug crizotinib, in appropriately stratified patients.Significance: E-cadherin defects are common in breast cancer but are currently not targeted with a precision medicine approach. Our preclinical data indicate that licensed ROS1 inhibitors, including crizotinib, should be repurposed to target E-cadherin–defective breast cancers, thus providing the rationale for the assessment of these agents in molecularly stratified phase II clinical trials. Cancer Discov; 8(4); 498–515. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 371

Supplementary Table S1. KCL TNBC and breast cell line cohort characteristics. Supplementary Table S2. Categories of genomic segment used as the basis for measuring SCINS. Supplementary Table S3. Genes expressed in a SAM of different SCINS clusters. Supplementary Table S4. Gene expression signatures of genomic instability.

Supplementary Tables

SWEAVE document. R code for SCINS analysis.

Supplementary Figures

Supplementary Figure legends and methods

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.

Evaluation of FRalpha expression by tumor cell lines

Table of Patient Characteristics

Cancer cells tend to metastasize first to tumor-draining lymph nodes, but the mechanisms mediating cancer cell invasion into the lymphatic vasculature remain little understood. Here, we show that in the human breast tumor microenvironment (TME), the presence of increased numbers of RORγt+ group 3 innate lymphoid cells (ILC3) correlates with an increased likelihood of lymph node metastasis. In a preclinical mouse model of breast cancer, CCL21-mediated recruitment of ILC3 to tumors stimulated the production of the CXCL13 by TME stromal cells, which in turn promoted ILC3–stromal interactions and production of the cancer cell motile factor RANKL. Depleting ILC3 or neutralizing CCL21, CXCL13, or RANKL was sufficient to decrease lymph node metastasis. Our findings establish a role for RORγt+ILC3 in promoting lymphatic metastasis by modulating the local chemokine milieu of cancer cells in the TME. Cancer Res; 77(5); 1083–96. ©2017 AACR.

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

Supplementary Figures and legends

IgE antibodies are key mediators of antiparasitic immune responses, but their potential for cancer treatment via antibody-dependent cell-mediated cytotoxicity (ADCC) has been little studied. Recently, tumor antigen–specific IgEs were reported to restrict cancer cell growth by engaging high-affinity Fc receptors on monocytes and macrophages; however, the underlying therapeutic mechanisms were undefined and in vivo proof of concept was limited. Here, an immunocompetent rat model was designed to recapitulate the human IgE-Fcϵ receptor system for cancer studies. We also generated rat IgE and IgG mAbs specific for the folate receptor (FRα), which is expressed widely on human ovarian tumors, along with a syngeneic rat tumor model expressing human FRα. Compared with IgG, anti-FRα IgE reduced lung metastases. This effect was associated with increased intratumoral infiltration by TNFα+ and CD80+ macrophages plus elevated TNFα and the macrophage chemoattractant MCP-1 in lung bronchoalveolar lavage fluid. Increased levels of TNFα and MCP-1 correlated with IgE-mediated tumor cytotoxicity by human monocytes and with longer patient survival in clinical specimens of ovarian cancer. Monocytes responded to IgE but not IgG exposure by upregulating TNFα, which in turn induced MCP-1 production by monocytes and tumor cells to promote a monocyte chemotactic response. Conversely, blocking TNFα receptor signaling abrogated induction of MCP-1, implicating it in the antitumor effects of IgE. Overall, these findings show how antitumor IgE reprograms monocytes and macrophages in the tumor microenvironment, encouraging the clinical use of IgE antibody technology to attack cancer beyond the present exclusive reliance on IgG. Cancer Res; 77(5); 1127–41. ©2017 AACR.

Supplementary Methods described

Time lapse microscopy experiment of sorted NKp46-ILC3 cells (CD3-, CD11c-, B220, NKp46-, CD127+, CD90.2+) co-cultured with MSC cells as described in Figure 2B. Low magnification (Ã-10) video shows the general clustering pattern of NKp46-ILC3 cells around the MSC cells over a duration of 10 hours.

Purpose: Highly aggressive triple-negative breast cancers (TNBCs) lack validated therapeutic targets and have high risk of metastatic disease. Folate receptor alpha (FRα) is a central mediator of cell growth regulation that could serve as an important target for cancer therapy.Experimental Design: We evaluated FRα expression in breast cancers by genomic (n = 3,414) and IHC (n = 323) analyses and its association with clinical parameters and outcomes. We measured the functional contributions of FRα in TNBC biology by RNA interference and the antitumor functions of an antibody recognizing FRα (MOv18-IgG1), in vitro, and in human TNBC xenograft models.Results: FRα is overexpressed in significant proportions of aggressive basal like/TNBC tumors, and in postneoadjuvant chemotherapy–residual disease associated with a high risk of relapse. Expression is associated with worse overall survival. TNBCs show dysregulated expression of thymidylate synthase, folate hydrolase 1, and methylenetetrahydrofolate reductase, involved in folate metabolism. RNA interference to deplete FRα decreased Src and ERK signaling and resulted in reduction of cell growth. An anti-FRα antibody (MOv18-IgG1) conjugated with a Src inhibitor significantly restricted TNBC xenograft growth. Moreover, MOv18-IgG1 triggered immune-dependent cancer cell death in vitro by human volunteer and breast cancer patient immune cells, and significantly restricted orthotopic and patient-derived xenograft growth.Conclusions: FRα is overexpressed in high-grade TNBC and postchemotherapy residual tumors. It participates in cancer cell signaling and presents a promising target for therapeutic strategies such as ADCs, or passive immunotherapy priming Fc-mediated antitumor immune cell responses. Clin Cancer Res; 24(20); 5098–111. ©2018 AACR.

Figure S1, Related to Figure 1 FACS and immunofluorescence validation of Lin-CD127+CD90.2+NKP46- gated ILC3 cells Figure S2, Related to Figure 2 ELISA quantification of CXCL13 and CCL21 chemokines levels in conditioned media (CM) obtained from the bone marrow derived MSC cell line (HS-5) and from the breast cancer cell line 4T1.2 cell line is shown. Figure S3, Related to Figure 2 Proliferation assay of ILC3 cells and ELISA quantification of CXCL13 and CCL21 from siRNA transfected MSC cells. Figure S4, Related to Figure 4 CXCR5 expression on MSC cells. Figure S5, Related to Figure 5 Identification of a RORγT+CD127+CD3- ILC3 cells within the tertiary lymphoid structures within breast cancers. Figure S6, Related to Figure 5 Expression of lymphoid chemokine and chemokine receptor genes in breast cancer datasets. Table S1, Related to Figure 5 Table S1: Clinico-pathological characteristics for the METABRIC sample.

Description of additional experiments and procedures carried out in this study

Supplementary Figure Legends, Materials and Methods

Supplementary figures

Supplementary Figure 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

Supplementary Table S1. List of genes with an absolute copy number of h >=5 for >=25% of TNBCs.

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 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.

Systemic immune responses in lymph nodes (LN) convey significant prognostic value for breast cancer patients, which can inform disease progression and optimal treatment management. However, have, so far, not been assessed in large patient cohorts. We have previously shown that morphological alterations in axillary LNs, namely the formation of germinal centres (GCs) in cancer-free LNs, add prognostic value to tumour infiltrating lymphocytes (TILs) in triple-negative breast cancer patients (TNBC) for the development of distant metastasis. Extending manual assessment of LNs beyond the detection of cancer requires the integration of robust deep learning pipelines into the digital pathology workflow. Here, we propose a supervised multiscale deep learning framework named smuLymphNet to capture and quantify GCs and sinuses within LNs from digitised Haematoxylin and Eosin-stained (H&E) whole slide images (WSIs) and show good concordance compared with an inter-pathologist Dice coefficient of manual annotations from four pathologists. The smuLymphNet framework consists of (i) a detection algorithm to determine the boundaries of each LN section on the WSI, using an Otsu-based thresholding method and contouring algorithm; (ii) a supervised multiscale deep learning module for the segmentation of GCs and sinuses; and (iii) quantification of the number, size, and shape of the predicted features. We applied smuLymphNet to a total of 1,800 H&E-stained WSI of >4,000 cancer-free and involved LNs from a retrospectively collected breast cancer cohort collected at Guy’s Hospital (London, UK) from 177 patients (122 N+) enriched for the triple-negative phenotype. A subset of 114 WSI and five breast cancer LN WSIs from each Barts Hospital (London, UK) and Tianjin University Hospital (Tianjin, China) were used to train and evaluate the supervised deep learning module. For training Fully Convolutional Networks (FCNs), WSIs manually annotated for both GCs and sinuses formed a ground-truth set and three FCNs were implemented: (i) a standard U-Net architecture; (ii) a U-Net model with an attention gate mechanism; and (iii) a multiscale-U-Net network (MS U-Net) that encodes, in parallel, a feature representation of the image at multiple resolutions. The MS U-Net achieved the best performance with an average dice score of 0.86 for GCs and 0.74 for sinuses. In comparison, the average dice score amongst four pathologists assessing 24 LN WSI for GCs and sinuses was 0.67 and 0.61, respectively, demonstrating the robustness of the smuLymphNet framework. To establish associations between morphometric immune features and patients’ outcomes, we assessed smuLymphNet captured GCs and sinuses from 686 WSIs from 96 TNBC patients with extensive longitudinal outcome data. We found significant morphological differences in involved and cancer-free LNs between N0 and N+ patients, with the latter displaying larger GCs with more irregular shapes, especially in their involved LNs. Moreover, in alignment with our previously published studies, our multiscale smuLymphNet framework recapitulated and extended the prognostic value of the assessment of GC formation in TNBC N0 patients. We further revealed, for the first time, the prognostic significance of the intranodal lymphatic sinuses when measured in their totality in involved LNs, and the association of alterations in subcapsular sinus areas with superior distant metastasis-free survival in cancer-free and involved LNs in TNBC N+ patients. In summary, smuLymphNet presents a robust multiscale deep learning framework to automatically detect, localise and quantify histopathological immune features in WSI of LNs. By applying smuLymphNet to LNs of TNBC patients from clinical trials, and thereby further evaluating its clinical utility, smuLymphNet could be implemented into the diagnostic digital pathology workflow and, as such, aid in informing on a patient’s disease trajectory. Citation Format: Gregory Verghese, Mengyuan Li, Fangfang Liu, Amit Lohan, Nikhil Cherian, Patrycja Gazinska, Aekta Shah, Aasiyah Oozeer, Cheryl Gillett, Elena Alberts, Thomas Hardiman, Roberto Salgado, Samantha Jones, Louise Jones, Selvam Thavaraj, Sarah E. Pinder, Swapni Rane, Amit Sethi, Anita Grigoriadis. Multiscale Deep Learning framework to capture systemic immune features in lymph nodes predictive of triple negative breast cancer outcome [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 P5-01-01.

Purpose: To assess the frequency and prognostic relevance of germinal centres (GC) in cancer-free axillary lymph nodes (ALNs), in relation to stromal tumour-infiltrating lymphocytes (sTILs) and the presence of tertiary lymphoid structures (TLS) in the primary carcinoma, in LN-positive Hormone Receptor (HR)-negative invasive breast cancer patients. Patients and methods: A cohort of 161 patients with HR-negative invasive breast cancer of no special type (NST) and LN-positive status treated between 2005-10 at Tianjin Medical University (China) was identified. sTILs and TLS at the primary tumour site and GC in 2,841 involved and cancer-free ALNs were evaluated on H&E stained sections. Markers were tested for prognostic value for invasive Disease-Free Survival (iDFS), distant Disease-Free Survival (dDFS) and Overall Survival (OS), using Cox regression models adjusted for clinico-pathological factors. Results: Among the 161 HR-negative breast cancers, 47% (n=75) had >=20% sTILs and 24% (n=38) peritumoural TLS. 75% (121/161) and 76% (122/161), respectively, displayed GCs in their cancer-free and involved ALNs. Significantly higher numbers of GCs were seen in both cancer-free and involved ALNs when the primary tumours showed >=20% sTILs. The presence of TLS was significantly associated with increased GC numbers in involved but to a lesser extend in cancer-free ALNs (Kruskal-Wallis rank sum test, p Using an iterative process to determine an optimal cut off point by a minimal P value approach, a non-monotonic relationship between the frequency of GC in cancer-free ALNs and all endpoints was observed. Patients with >2 GC but less than the top 5% (= 62 GC) in cancer-free ALNs showed improved iDFS, dDFS and OS, whilst patients with 62 GC across all assessed cancer-free ALNs had poorer iDFS, dDFS and OS (see Table). In the multivariate models, the frequency of GC in cancer-free ALNs added independent prognostic information for all endpoints across all patients. In patients with >=20% sTILS, cancer-free ALNs within the top 5% for GC remained associated with a worse dDFS and iDFS (see Table). Cancer-free ALNs with Five-year iDFS, dDFS and OS in patients with 2 GC whose five-year iDFS, dDFS and OS were 65%, 65% and 69%, respectively. Conclusions: The prognostic importance of GC assessment in cancer-free ALNs in women with LN-positive HR-negative breast cancers is demonstrated. A better outcome in patients with GC formation in their cancer-free ALNs, despite low sTILs in the primary carcinoma, may suggest a systemic anticancer immune response. High sTILs but with extreme GC formation in the cancer-free ALNs could potentially reflect an overdriving but less effective immune response. The assessment of the combination of primary and nodal immune response in HR-negative breast cancers is imperative in these high-risk patients. Table 1. Univariate and Multivariate Cox Regression Analysis of Outcome by GC in Cancer-Free ALNsiDFSUnivariateAll cases=20% sTILTotal GCs number - binnedModel PHRCIModel PHRCIModel PHRCIGC624.631.75 - 12.41.930.26 - 14.5612.793.34 - 48.97MultivariateCorrected for: pNstage, sTILS & TLSCorrected for: pTstage & TLSCorrected for: pNstageTotal GCs number - binnedCovariate PModel PHRCICovariate PModel PHRCICovariate PModel PHRCIGC624.15E-059.583.25 - 28.201.94E-014.070.49 - 33.876.62E-038.861.83 - 42.82dDFSUnivariateAll cases=20% sTILTotal GCs number - binnedModel PHRCIModel PHRCIModel PHRCIGC625.872.14 - 16.082.490.32 - 19.0718.434.04 - 84.03MultivariateCorrected for: pTstage, pNstage, sTILS & TLSCorrected for: Age, pTstage, TLSCorrected for: pNstageTotal GCs number - binnedCovariate PModel PHRCICovariate PModel PHRCICovariate PModel PHRCIGC622.49E-0615.304.91 - 47.635.19E-028.610.98 - 75.553.11E-0313.302.39 - 73.89OSUnivariateAll cases=20% sTILTotal GCs number - binnedModel PHRCIModel PHRCIModel PHRCIGC624.131.18 - 14.42.570.33 - 19.8311.891.65 - 85.45MultivariateCorrected for: Age, pTstage, pNstage, sTILS & TLSCorrected for: Age & TLSCorrected for: pNstage, LVI & TLSTotal GCs number - binnedCovariate PModel PHRCICovariate PModel PHRCICovariate PModel PHRCIGC628.62E-0516.064.02 - 64.247.01E-027.050.85 - 58.342.80E-012.970.41 - 21.43*too few events Citation Format: Fangfang Liu, Thomas Hardiman, Patrycja Gazinska, Jelmar Quist, Sarah Pinder, Anita Grigoriadis. The formation of GCs in cancer-free ALNs, a non-monotonic prognostic factor in HR-negative invasive breast cancer patients [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS6-36.

Background In the TNT trial of triple negative breast cancer (NCT00532727), germline BRCA1/2 mutations were present in 28% of carboplatin responders. We assessed quantitative measures of structural chromosomal instability (CIN) to identify a wider patient subgroup within TNT with preferential benefit from carboplatin over docetaxel. Patients and methods Copy number aberrations (CNAs) were established from 135 formalin-fixed paraffin-embedded primary carcinomas using Illumina OmniExpress SNP-arrays. Seven published [allelic imbalanced CNA (AiCNA); allelic balanced CNA (AbCNA); copy number neutral loss of heterozygosity (CnLOH); number of telomeric allelic imbalances (NtAI); BRCA1-like status; percentage of genome altered (PGA); homologous recombination deficiency (HRD) scores] and two novel [Shannon diversity index (SI); high-level amplifications (HLAMP)] CIN-measurements were derived. HLAMP was defined based on the presence of at least one of the top 5% amplified cytobands located on 1q, 8q and 10p. Continuous CIN-measurements were divided into tertiles. All nine CIN-measurements were used to analyse objective response rate (ORR) and progression-free survival (PFS). Results Patients with tumours without HLAMP had a numerically higher ORR and significantly longer PFS in the carboplatin (C) than in the docetaxel (D) arm [56% (C) versus 29% (D), PHLAMP,quiet = 0.085; PFS 6.1 months (C) versus 4.1 months (D), Pinteraction/HLAMP = 0.047]. In the carboplatin arm, patients with tumours showing intermediate telomeric NtAI and AiCNA had higher ORR [54% (C) versus 20% (D), PNtAI,intermediate = 0.03; 62% (C) versus 33% (D), PAiCNA,intermediate = 0.076]. Patients with high AiCNA and PGA had shorter PFS in the carboplatin arm [3.4 months (high) versus 5.7 months (low/intermediate); and 3.8 months (high) versus 5.6 months (low/intermediate), respectively; Pinteraction/AiCNA = 0.027, Padj.interaction/AiCNA = 0.125 and Pinteraction/PGA = 0.053, Padj.interaction/PGA = 0.176], whilst no difference was observed in the docetaxel arm. Conclusions Patients with tumours lacking HLAMP and demonstrating intermediate CIN-measurements formed a subgroup benefitting from carboplatin relative to docetaxel treatment within the TNT trial. This suggests a complex and paradoxical relationship between the extent of genomic instability in primary tumours and treatment response in the metastatic setting., Highlights • Patients with intermediate levels of allelic imbalanced CNAs show a better response rate to carboplatin in TNT. • The lack of amplifications on 1q, 8q and 10p is associated with a superior carboplatin response in TNT. • The relation between chromosomal instability in primary tumours and carboplatin response in advanced settings is non-linear.