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Purpose: The local immune infiltrate's influence on tumor progression may be closely linked to tumor-intrinsic factors. The study aimed to investigate whether integrating immunologic and tumor-intrinsic factors can identify patients from a low-risk cohort who may be candidates for radiotherapy (RT) de-escalation. Experimental Design: The SweBCG91RT trial included 1,178 patients with stage I to IIA breast cancer, randomized to breast-conserving surgery with or without adjuvant RT, and followed for a median of 15.2 years. We trained two models designed to capture immunologic activity and immunomodulatory tumor-intrinsic qualities, respectively. We then analyzed if combining these two variables could further stratify tumors, allowing for identifying a subgroup where RT de-escalation is feasible, despite clinical indicators of a high risk of ipsilateral breast tumor recurrence (IBTR). Results: The prognostic effect of the immunologic model could be predicted by the tumor-intrinsic model (Pinteraction = 0.01). By integrating measurements of the immunologic- and tumor-intrinsic models, patients who benefited from an active immune infiltrate could be identified. These patients benefited from standard RT (HR, 0.28; 95% CI, 0.09–0.85; P = 0.025) and had a 5.4% 10-year incidence of IBTR after irradiation despite high-risk genomic indicators and a low frequency of systemic therapy. In contrast, high-risk tumors without an immune infiltrate had a high 10-year incidence of IBTR despite RT treatment (19.5%; 95% CI, 12.2–30.3). Conclusions: Integrating tumor-intrinsic and immunologic factors may identify immunogenic tumors in early-stage breast cancer populations dominated by ER-positive tumors. Patients who benefit from an activated immune infiltrate may be candidates for RT de-escalation.

Background: The de novo drug development process is expensive and challenging, with a high risk of failure. Drug repurposing can ideally identify novel therapeutic indications for FDA-approved drugs with pre-existing pre-clinical and clinical evidence. Both aspirin and tamoxifen drugs are good examples of successful drug repurposing in oncology. Although proteasome inhibitors such as bortezomib and carfilzomib are currently only used to treat multiple myeloma and basal cell lymphoma, we and others have shown that triple-negative breast cancer (TNBC) is particularly sensitive to proteasome inhibition. TNBC is an aggressive form of breast cancer with an urgent need for novel treatment options. Here, we evaluate the potency of proteasome inhibitors and other clinically relevant chemotherapeutic agents on TNBC cell lines. Methods: We performed a high-throughput drug sensitivity screen with eight cell lines representing the four TNBC subtypes (basal-like 1: HCC70 and MDA-MB-468; basal-like 2: HCC1806 and MDA-MB-436; mesenchymal-like: BT-549 and HCC38; luminal androgen receptor: CAL-148 and MDA-MB-435) and MCF-7 as control (estrogen and progesterone receptor-positive) exposed to 18 drugs (11 proteasome inhibitors, 2 mitosis inhibitors, 2 topoisomerase inhibitors, and 3 platinum agents) for 24 hours. Drug potency was determined using the IC50, GR50, GRmax drug metrics. IDACombo was then used to predict efficacious drug combinations, followed by calculation of synergistic drug combinations with SynergyFinder. Results: TNBC cell lines were generally more sensitive to proteasome inhibitors with significantly reduced cell viability than clinically relevant drugs, e.g. paclitaxel. Although the potency of different proteasome inhibitors varied, the most potent proteasome inhibitors included bortezomib, carfilzomib, delanzomib, epoxomicin, and MLN-2238. According to the GR50 values, HCC38 (range, 8.2-382.7 nM) and MDA-MB-468 (range, 10.8-110.6 nM) were most sensitive to proteasome inhibition, whereas the least sensitive TNBC cell lines were HCC1806 (range, 289.9-Inf nM) and BT-549 (range, 101.0-Inf nM). Using the drug sensitivity screening results for single drugs, IDACombo predicted potent drug combinations for different combinations of bortezomib, carboplatin, carfilzomib, delanzomib, docetaxel, doxorubicin, epirubicin, epoxomicin, MLN-2238, MLN-9708, and nedaplatin. Conclusions: In summary, some proteasome inhibitors (e.g. bortezomib) had a substantial impact on TNBC cell survival. These findings indicate that proteasome inhibitors, together with other forms of chemotherapy, may be further explored as a novel complement treatment for TNBC. Citation Format: Peter Larsson, Daniella Pettersson, Maxim Olsson, Eva Forssell-Aronsson, Anikó Kovács, Per Karlsson, Khalil Helou, Toshima Z. Parris. Repurposing proteasome inhibitors for improved treatment of triple-negative breast cancer [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 P4-08-17.

Ki67 was evaluated by a board-certified pathologist on TMAs.

Gene sets from the molecular signatures database were used to create a model measuring immunological activity and immunomodulatory tumor-intrinsic factors. These were merged to create a model considering the interaction between the antitumoral immune response and tumor-intrinsic immunomodulatory factors.

Purpose:The local immune infiltrate's influence on tumor progression may be closely linked to tumor-intrinsic factors. The study aimed to investigate whether integrating immunologic and tumor-intrinsic factors can identify patients from a low-risk cohort who may be candidates for radiotherapy (RT) de-escalation.Experimental Design:The SweBCG91RT trial included 1,178 patients with stage I to IIA breast cancer, randomized to breast-conserving surgery with or without adjuvant RT, and followed for a median of 15.2 years. We trained two models designed to capture immunologic activity and immunomodulatory tumor-intrinsic qualities, respectively. We then analyzed if combining these two variables could further stratify tumors, allowing for identifying a subgroup where RT de-escalation is feasible, despite clinical indicators of a high risk of ipsilateral breast tumor recurrence (IBTR).Results:The prognostic effect of the immunologic model could be predicted by the tumor-intrinsic model (Pinteraction = 0.01). By integrating measurements of the immunologic- and tumor-intrinsic models, patients who benefited from an active immune infiltrate could be identified. These patients benefited from standard RT (HR, 0.28; 95% CI, 0.09–0.85; P = 0.025) and had a 5.4% 10-year incidence of IBTR after irradiation despite high-risk genomic indicators and a low frequency of systemic therapy. In contrast, high-risk tumors without an immune infiltrate had a high 10-year incidence of IBTR despite RT treatment (19.5%; 95% CI, 12.2–30.3).Conclusions:Integrating tumor-intrinsic and immunologic factors may identify immunogenic tumors in early-stage breast cancer populations dominated by ER-positive tumors. Patients who benefit from an activated immune infiltrate may be candidates for RT de-escalation.

Immunescore (A), developed in this manuscript, showed a stronger correlation with tumor-infiltrating lymphocytes than other developed methods (B and C) to quantify the degree of immune infiltration.

The interaction between the Integrated model and RT was significant in an unadjusted analysis (p=0.004) and an analysis adjusted for histological grade, age, ER status, and tumor size (p=0.008). The figure shows the predicted 10-year cumulative incidence of IBTR with 95% confidence intervals based on Integrated model score stratified by RT, based on Royston-Parmar models with estimates from Table S10. The stpm2 macro for Stata was used for the calculations. The dotted vertical lines represent percentiles of the Integrated score.

Supplementary Table S1 Demographics of the training cohortsSupplementary Table S2 Immunscore modelSupplementary Table S3 Proliferative Index ModelSupplementary Table S4 Immunescore:Proliferative Index

Supplementary MethodsTable S5. Clinical characteristics of the Sjöström and Servant cohortsTable S6. Comparison of included versus excluded patientsTable S7. 10-year follow-up for local recurrence (IBTR). Flexible parametric survival analysis with Royston-Parmar (RP) regression models used in Figures 2 and 3Table S8. 10-year follow-up for local recurrence (IBTR). Flexible parametric survival analysis with Royston-Parmar (RP) regression models with adjustment for other covariatesTable S9. Absolute number of events within different tertiles of Immunescore and Proliferative Index and depending on RT treatmentTable S10. Unadjusted and adjusted analysis of the interaction between the Integrated model and RT in the SweBCG91RT cohortTable S11. Distribution of subtypes in the tertiles of the Integrated score among high-risk patients

Protocol for original SweBCG91RT trial

The tumor-intrinsic model, Proliferative Index, could predict the prognostic effect from the immunological model, Immunescore. With high Proliferative Index values, Immunescore was strongly prognostic. However, with low Proliferative Index values, trends toward the opposite were observed.

Study protocol

Supplementary Data from Clinical Implications of Gene Dosage and Gene Expression Patterns in Diploid Breast Carcinoma

Supplementary Table S5 from Clinical Implications of Gene Dosage and Gene Expression Patterns in Diploid Breast Carcinoma

Supplementary Table S1 from Clinical Implications of Gene Dosage and Gene Expression Patterns in Diploid Breast Carcinoma

Supplementary Table S6 from Clinical Implications of Gene Dosage and Gene Expression Patterns in Diploid Breast Carcinoma

Supplementary Table S3 from Clinical Implications of Gene Dosage and Gene Expression Patterns in Diploid Breast Carcinoma

Purpose: Deregulation of key cellular pathways is fundamental for the survival and expansion of neoplastic cells. In cancer, regulation of gene transcription can be mediated in a variety of ways. The purpose of this study was to assess the impact of gene dosage on gene expression patterns and the effect of other mechanisms on transcriptional levels, and to associate these genomic changes with clinicopathologic parameters.Experimental Design: We screened 97 invasive diploid breast tumors for DNA copy number alterations and changes in transcriptional levels using array comparative genomic hybridization and expression microarrays, respectively.Results: The integrative analysis identified an increase in the overall number of genetic alterations during tumor progression and 15 specific genomic regions with aberrant DNA copy numbers in at least 25% of the patient population, i.e., 1q22, 1q22-q23.1, 1q25.3, 1q32.1, 1q32.1-q32.2, 8q21.2-q21.3, 8q22.3, 8q24.3, and 16p11.2 were recurrently gained, whereas 11q25, 16q21, 16q23.3, and 17p12 were frequently lost (P < 0.01). An examination of the expression patterns of genes mapping within the detected genetic aberrations identified 47 unique genes and 1 Unigene cluster significantly correlated between the DNA and relative mRNA levels. In addition, more malignant tumors with normal gene dosage levels displayed a recurrent overexpression of UBE2C, S100A8, and CBX2, and downregulation of LOC389033, STC2, DNALI1, SCUBE2, NME5, SUSD3, SERPINA11, AZGP1, and PIP.Conclusions: Taken together, our findings suggest that the dysregulated genes identified here are critical for breast cancer initiation and progression, and could be used as novel therapeutic targets for drug development to complement classical clinicopathologic features. Clin Cancer Res; 16(15); 3860–74. ©2010 AACR.

Purpose:Tumor-infiltrating immune cells play a key role in tumor progression. The purpose of this study was to analyze whether the immune infiltrate predicts benefit from postoperative radiotherapy in a large randomized breast cancer radiotherapy trial.Experimental Design:In the SweBCG91RT trial, patients with stage I and II breast cancer were randomized to breast-conserving surgery (BCS) and postoperative radiotherapy or to BCS only and followed for a median time of 15.2 years. The primary tumor immune infiltrate was quantified through two independent methods: IHC and gene expression profiling. For IHC analyses, the absolute stromal area occupied by CD8+ T cells and FOXP3+ T cells, respectively, was used to define the immune infiltrate. For gene expression analyses, immune cells found to be prognostic in independent datasets were pooled into two groups consisting of antitumoral and protumoral immune cells, respectively.Results:An antitumoral immune response in the primary tumor was associated with a reduced risk of breast cancer recurrence and predicted less benefit from adjuvant radiotherapy. The interaction between radiotherapy and immune phenotype was significant for any recurrence in both the IHC and gene expression analyses (P = 0.039 and P = 0.035) and was also significant for ipsilateral breast tumor recurrence in the gene expression analyses (P = 0.025).Conclusions:Patients with an antitumoral immune infiltrate in the primary tumor have a reduced risk of any recurrence and may derive less benefit from adjuvant radiotherapy. These results may impact decisions regarding postoperative radiotherapy in early breast cancer.

Supplementary Table S4 from Clinical Implications of Gene Dosage and Gene Expression Patterns in Diploid Breast Carcinoma

Progression to metastasis is the proximal cause of most cancer-related mortality. Yet much remains to be understood about what determines the spread of tumor cells. This paper describes a novel pathway in breast cancer that regulates epithelial-to-mesenchymal transition (EMT), motility, and invasiveness. We identify two transcription factors, nuclear factor 1-C2 (NF1-C2) and Forkhead box F1 (FoxF1), downstream of prolactin/nuclear Janus-activated kinase 2, with opposite effects on these processes. We show that NF1-C2 is lost during mammary tumor progression and is almost invariably absent from lymph node metastases. NF1-C2 levels in primary tumors correlate with better patient survival. Manipulation of NF1-C2 levels by expression of a stabilized version or using small interfering RNA showed that NF1-C2 counteracts EMT, motility, invasiveness, and tumor growth. FoxF1 was found to be a direct repressed target of NF1-C2. We provide the first evidence for a role of FoxF1 in cancer and in the regulation of EMT in cells of epithelial origin. Overexpression of FoxF1 was associated with a mesenchymal phenotype, increased invasiveness in vitro, and enhanced growth of breast carcinoma xenografts in nude mice. The relevance of these findings is strengthened by the correlation between FoxF1 expression and a mesenchymal phenoype in breast cancer cell isolates, consistent with the interpretation that FoxF1 promotes invasion and metastasis. Cancer Res; 70(5); 2020–9

Supplementary Table from Nuclear Janus-Activated Kinase 2/Nuclear Factor 1-C2 Suppresses Tumorigenesis and Epithelial-to-Mesenchymal Transition by Repressing Forkhead Box F1

Supplementary Figure 1 from Nuclear Janus-Activated Kinase 2/Nuclear Factor 1-C2 Suppresses Tumorigenesis and Epithelial-to-Mesenchymal Transition by Repressing Forkhead Box F1

Supplementary Figure Legend from Nuclear Janus-Activated Kinase 2/Nuclear Factor 1-C2 Suppresses Tumorigenesis and Epithelial-to-Mesenchymal Transition by Repressing Forkhead Box F1

Supplementary Movie 1 from Nuclear Janus-Activated Kinase 2/Nuclear Factor 1-C2 Suppresses Tumorigenesis and Epithelial-to-Mesenchymal Transition by Repressing Forkhead Box F1

Supplementary Movie Legends 1-2 from Nuclear Janus-Activated Kinase 2/Nuclear Factor 1-C2 Suppresses Tumorigenesis and Epithelial-to-Mesenchymal Transition by Repressing Forkhead Box F1

Supplementary Methods from Nuclear Janus-Activated Kinase 2/Nuclear Factor 1-C2 Suppresses Tumorigenesis and Epithelial-to-Mesenchymal Transition by Repressing Forkhead Box F1

Supplementary Movie 2 from Nuclear Janus-Activated Kinase 2/Nuclear Factor 1-C2 Suppresses Tumorigenesis and Epithelial-to-Mesenchymal Transition by Repressing Forkhead Box F1

Supplementary Table Legend from Nuclear Janus-Activated Kinase 2/Nuclear Factor 1-C2 Suppresses Tumorigenesis and Epithelial-to-Mesenchymal Transition by Repressing Forkhead Box F1

Purpose: The effect of postoperative radiotherapy (RT) based on the immune phenotype of tumor-infiltrating lymphocytes (TILs) has not been investigated. The purpose of this study was to analyze how the balance between CD8+ T cells and T regulatory cells (FOXP3+), which have opposite effects on the anti-tumoral defense, affects the risk of ipsilateral breast tumor recurrence (IBTR) and of any recurrence as well as the interaction with RT in a large randomized RT trial. Methods: In the SweBCG91RT trial, patients with breast cancer stage I and II were randomized to breast conserving surgery (BCS) and postoperative RT or BCS only and followed for a median time of 15.2 years. Tumor blocks were retrieved and stromal TILs were assessed through hematoxylin-eosin stained slides. CD8+ T cells and T regulatory cells were evaluated through staining for CD8 and FOXP3 and the percentage of stroma occupied by CD8+ T cells and T regulatory cells respectively was then calculated. Cutoffs at 5% and 2.5% were used to define high levels of CD8+ T cells (CD8High) and T regulatory cells (FOXP3High), respectively. In total, 943 patients were analyzed. Results: Among patients who did not receive RT, an increased risk of IBTR and of any recurrence was seen in the CD8Low/FOXP3Low (HR 2.64, CI95% 1.26-5.56, p=0.010 and HR 2.52, CI95% 1.34-4.77, p=0.004, respectively) and CD8High/FOXP3High (HR 1.94, CI95% 0.81-4.63, p=0.135 and HR 2.76, CI95% 1.36-5.57, p=0.005, respectively) groups compared to CD8High/FOXP3Low (HR 1.0) in multivariable analyses including grade and age. The effect of RT on risk of IBTR was more pronounced in the groups with CD8Low/FOXP3Low (HR 0.37, CI95% 0.24-0.57, p Conclusions: Our findings suggest that patients with a favorable immune phenotype (CD8High/FOXP3Low) may not derive any benefit from adjuvant RT which could be explained by an interaction through which RT may suppress an activated immune response. These results may have an impact on decisions regarding postoperative RT in early breast cancer. Citation Format: Axel Stenmark Tullberg, Henri AJ Puttonen, Erik Holmberg, Dan Lundstedt, Fredrika Killander, Emma Niméus, Anikó Kóvacs, Per Karlsson. A high ratio of CD8/FOXP3 predicts an unfavorable response to postoperative radiotherapy after breast-conserving surgery: Results from the randomized SweBCG91RT trial [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr PD6-10.

The site of relapse is known to be a strong prognostic factor of survival from metastatic breast cancer. The liver is the third most frequent site affected and patients with hepatic metastases have an inferior prognosis relative to patients with bone and lung metastases. Predicting the likelihood of liver metastasis after adjuvant therapy of primary breast cancer is challenging. The aim of this study was to identify and characterize genes associated with breast cancer liver metastases. We thus performed global transcriptional profiling of 120 metastatic lesions from different anatomical sites from breast cancer patients enrolled in a randomized clinical trial1 and found that while the expression of other matrix remodelling and tight-junction molecules was down-regulated in liver metastases, Claudin-2 (CLDN2) was significantly up-regulated (p = 0.001). Furthermore, high expression of CLDN2 was also observed amongst patients diagnosed with a liver metastasis, irrespective of the specific site of the metastasis that was profiled, compared to patients without hepatic metastases (p = 0.001). We further investigated the prognostic significance of CLDN2 protein expression by immunohistochemical staining of primary tumours and matched lymph node metastases from a larger cohort of patients (n∼200) with metastatic breast cancer. We found CLDN2 to be significantly more frequently expressed in lymph node metastases compared to primary tumours (p = 0.013). Also, consistent with mRNA expression levels, more frequent expression of CLDN2 protein was observed in the primary tumours of patients who eventually developed a liver metastasis (p = 0.027). Interestingly, high expression of CLDN2 in the primary tumour was associated with a shorter time to recurrence (p = 0.032). Specifically, high expression of CLDN2 in the primary tumour was associated with a shorter progression to liver metastasis (p = 0.002). These data highlight a potential role of CLDN2 in the development and prognosis of breast cancer liver metastases. 1. Hatschek, T., et al. Individually tailored treatment with epirubicin and paclitaxel with or without capecitabine as first-line chemotherapy in metastatic breast cancer: a randomized multicenter trial. Breast Cancer Res Treat. 2012, 131(3):939–47. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-05-07.

Background: Breast cancer molecular subtypes preferentially metastasize to specific organs, but the underlying molecular mechanisms are not well understood. While several studies have attempted to delineate these mechanisms by making predictions from primary tumours, the likelihood of missing further relevant biological differences which may be enriched or distinctively present in the metastases cannot be overlooked. In addition, survival after relapse is influenced by the anatomical location of the metastasis, with visceral involvement associated with the poorest survival, suggesting that differences in biology and response to treatment may exist. However, the scarcity of clinical material from metastatic lesions has largely prevented detailed molecular characterization of breast cancer site-specific metastases. Material and Methods: We performed global gene expression profiling on 122 fine needle aspirates of metastatic lesions from different anatomical sites obtained from breast cancer patients treated within the Swedish randomized trial (TEX) of chemotherapy for metastatic breast cancer. Sub-classification was performed using unsupervised hierarchical clustering, while differentially expressed genes and modulated pathways in metastases from different sites and sub-classes were identified using Significance Analysis of Microarrays (SAM) analysis. Breast cancer survival after relapse was also compared between patients. Results: We confirmed the observation that breast cancer metastasis to visceral organs is associated with the poorest outcome. Molecularly, samples were sub-classified into groups with strong correlations with the previously described breast cancer intrinsic subtypes and also striking overlap with pathological characteristics of the primary tumour. Metastasis pairs from the same individual were more similar to each other than to independent metastatic lesions from other patients. More interestingly, liver metastases formed a unique sub-cluster which could be distinguished from the other sites by the expression of several genes (and pathways) involved in focal adhesion, matrix remodelling, and wnt signalling. Conclusions: Our data suggest that breast cancer molecular subtypes and primary tumour characteristics are to a large extent maintained in recurrences and further that liver metastases may be notably different and represent a distinct entity with a separate underlying biology which may hence require specific targeted therapeutic management. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3386. doi:1538-7445.AM2012-3386