Biomarkers of Response to Combinational Use of PD-1 Blockade and Anti-angiogenesis: An Analysis from a Phase II Trial in Advanced Triple-negative Breast Cancer Patients

Abstract Background: We recently reported the results of a phase II clinical trial that the combinational use of camrelizumab plus apatinib induced an objective response rate (ORR) at 43.3% in advanced triple-negative breast cancer (TNBC) patients. This study presents the analysis of potential tumor and blood biomarkers for the patients responded to the combinational therapy. Methods: Stromal tumor-infiltrating lymphocytes (TILs), CD8+ T cells and the protein expression of programmed death protein 1 (PD-1) and PD-L1 were evaluated in tumor samples collected before and after the treatment. Peripheral blood samples were collected before treatment, 2-weeks and 8-weeks after treatment. 59 cytokines/chemokines, growth factors, or checkpoint-related proteins, blood immune cell subpopulations were analyzed in the blood samples. The correlation between biomarkers and clinical outcomes including ORR, progression-free survival (PFS), and overall survival (OS) was analyzed. Results: Upon database lock, the ORR of 28 evaluable patients was 46.4%. An increase of tumor-infiltrating CD8+ T cells more than 15% during therapy was significantly associated with higher ORR (P=0.040). Patients with lower baseline plasma levels of HGF or IL-8 were more likely to respond to the combinational treatment (P=0.005 or 0.001, respectively), and showed a longer PFS and OS (HGF: PPFS<0.0001, POS<0.0001; IL-8: PPFS<0.0001, POS=0.009). Patients with a decrease of IL-8, or an increase of TIM-3 or CD152 during treatment responded more to the treatment (P=0.008, 0.040, or 0.014, respectively). Responders had a higher baseline CD4+ T cells and B cell proportions in blood than non-responders (P=0.002 and 0.030 respectively). Moreover, patients with higher baseline CD4+ T cells or B cells proportions in blood showed a longer PFS (P<0.001) or a longer OS (P=0.030) respectively. Conclusion: Higher baseline TILs or a greater increase of tumor-infiltrating CD8+ T cells during therapy, lower baseline plasma HGF/IL-8, a decrease of plasma IL-8, an increase of plasma TIM-3/CD152 during therapy, higher baseline CD4+ T cells or B cells proportion in blood are potential biomarkers for the combinational anti-angiogenesis and immunotherapy in advanced TNBC patients.

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Classification of triple-negative breast cancer based on immune cell infiltration

10.21203/rs.3.rs-43664/v1 ◽

2020 ◽

Author(s):

Wenji Shi ◽

Dan Wang ◽

Luz Angela Torres-de la Roche ◽

Rui Zhuo ◽

Rudy Leon De Wilde

Keyword(s):

Breast Cancer ◽

T Cells ◽

B Cells ◽

Triple Negative Breast Cancer ◽

Triple Negative ◽

Immune Cell ◽

Cell Infiltration ◽

Immune Cell Infiltration ◽

Immune Phenotype ◽

Cluster A

Abstract Background Although the classification system of triple-negative breast cancer(TNBC) has become more and more perfect, there is no report on the immune subtype of triple-negative breast cancer based on immune cell infiltration. Results According to immune infiltrating cells, data from 360 patients were divided into cluster A (subtype 1 and subtype 3) and cluster B (subtype 2; with poorly immune phenotype). Expression of memory B cells, naïve B cells, M0 macrophages, M1 macrophages, CD4 memory activated T cells, and CD4 naïve T cells were significantly higher in cluster A (P < 0.05). In contrast, the expression of M2 macrophages and resting mast cells were higher in cluster B (P < 0.05). GSVA results show that B cell receptor pathway and JAK-STAT pathway are activated and more frequently altered in cluster A (P < 0.05). mTOR pathway alterations usually appear in cluster B (P < 0.05). Compared with cluster A, the risk of recurrence in cluster B patients is significantly increased (P < 0.05). Conclusions This analysis of tumor microenvironment revealed the multifaceted nature of TNBC and its impact on patient prognosis, being recurrence more often in those with poorly immune phenotype. These results provide a reference for further exploration of the heterogeneity of TNBC.

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Epigenetic Quantification of Circulating Immune Cells in Peripheral Blood of Triple-Negative Breast Cancer patients

10.21203/rs.3.rs-508197/v1 ◽

2021 ◽

Author(s):

Mehdi Manoochehri ◽

Thomas Hielscher ◽

Nasim Borhani ◽

Clarissa Gerhäuser ◽

Olivia Fletcher ◽

...

Keyword(s):

Breast Cancer ◽

T Cells ◽

Triple Negative Breast Cancer ◽

Immune Cells ◽

Triple Negative ◽

Immune Cell ◽

Nk Cell ◽

Cell Type ◽

Cell Ratio ◽

Immune Cell Type

Abstract Background: A shift in the proportions of blood immune cells is a hallmark of cancer development. Here, we investigated whether methylation-derived immune cell type ratios and methylation-derived neutrophil-to-lymphocyte ratios (mdNLRs) are associated with triple-negative breast cancer (TNBC). Methods: Leukocyte subtype-specific un/methylated CpG sites were selected and methylation levels at these sites used as proxies for immune cell type proportions and mdNLR estimation in 231 TNBC cases and 231 age-matched controls. Data were validated using the Houseman deconvolution method. Additionally, the natural killer (NK) cell ratio was measured in a prospective sample set of 146 TNBC cases and 146 age-matched controls. Results: The mdNLRs were higher in TNBC cases compared with controls and associated with TNBC (odds ratio (OR) range (2.66-4.29), all Padj.<1e-04). A higher neutrophil ratio and lower ratios of NK cells, CD4+ T cells, CD8+ T cells, monocytes, and B cells were associated with TNBC. The strongest association was observed with decreased NK cell ratio (OR range (1.28-1.42), all Padj.<1e-04). The NK cell ratio was also significantly lower in pre-diagnostic samples of TNBC cases compared with controls (P=0.019).Conclusion: This immunomethylomic study shows that a shift in the ratios/proportions of leukocyte subtypes is associated with TNBC, with decreased NK cell showing the strongest association. These findings improve our knowledge of the role of the immune system in TNBC and point to the possibility of using NK cell level as a non-invasive molecular marker for TNBC risk assessment, early detection, and prevention.

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Efficacy and safety of camrelizumab combined with apatinib in advanced triple-negative breast cancer: an open-label phase II trial

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-000696 ◽

2020 ◽

Vol 8 (1) ◽

pp. e000696

Author(s):

Jieqiong Liu ◽

Qiang Liu ◽

Ying Li ◽

Qian Li ◽

Fengxi Su ◽

...

Keyword(s):

Breast Cancer ◽

Phase Ii ◽

Triple Negative Breast Cancer ◽

Triple Negative ◽

Phase Ii Trial ◽

Objective Response ◽

Therapeutic Effects ◽

Open Label ◽

Programmed Death ◽

Continuous Dosing

BackgroundPrevious trials showed that antiangiogenesis or anti-programmed death protein 1/programmed death ligand 1 (PD-1/PD-L1) monotherapy only showed marginal effect in triple-negative breast cancer (TNBC). Preclinical studies demonstrated that antiangiogenic therapy could sensitize breast cancer to PD-1/PD-L1 blockade via reprogramming tumor microenvironment. Combinational treatment of checkpoint blockade and antiangiogenesis for TNBC has not been reported.MethodsPatients with advanced TNBC with less than three lines of systemic therapy were enrolled in an open-label, non-comparative, two-arm, phase II trial at Sun Yat-sen Memorial Hospital. Camrelizumab (intravenously every 2 weeks) with apatinib orally at either continuous dosing (d1–d14) or intermittent dosing (d1–d7) was given until disease progression or unacceptable toxicities. Primary endpoint was objective response rate (ORR).ResultsFrom January 2018 to April 2019, 40 patients were enrolled, including 10 in the apatinib intermittent dosing cohort and 30 in the apatinib continuous dosing cohort. The ORR was 43.3% (13 of 30) in the continuous dosing cohort, while no objective response was observed in the intermittent dosing cohort. The disease control rate was 63.3% (19 of 30) in the apatinib continuous dosing cohort, and 40.0% (4 of 10) in the apatinib intermittent dosing cohort, respectively. The median progression-free survival (PFS) was 3.7 (95% CI 2.0 to 6.4) months and 1.9 (95% CI 1.8 to 3.7) months in the continuous dosing and intermittent dosing cohort, respectively. In the continuous dosing cohort, the median PFS of patients with partial response (8.3 months, 95% CI 5.9 to not reached) was significantly longer than that of patients with stable disease/progressive disease/not evaluable (2.0 months, 95% CI 1.7 to 3.0). The most common adverse events (AEs) included elevated aspartate aminotransferase/alanine aminotransferase and hand-foot syndrome. Overall, 26.7% and 20.0% of patients experienced grade ≥3 AEs in the continuous dosing and intermittent dosing cohort, respectively. In the continuous dosing cohort, a high percentage of baseline tumor-infiltrating lymphocytes (>10%) was associated with higher ORR and favorable PFS (p=0.029, 0.054, respectively).ConclusionsThe ORR by this chemo-free regimen was dramatically higher than previously reported ORR by anti-PD-1/PD-L1 antibody or apatinib monotherapy. Camrelizumab combined with apatinib demonstrated favorable therapeutic effects and a manageable safety profile in patients with advanced TNBC.Trial registration numberNCT03394287.

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Phase I/II trial of pembrolizumab in combination with binimetinib in unresectable locally advanced or metastatic triple negative breast cancer.

Journal of Clinical Oncology ◽

10.1200/jco.2018.36.5_suppl.tps17 ◽

2018 ◽

Vol 36 (5_suppl) ◽

pp. TPS17-TPS17 ◽

Cited By ~ 2

Author(s):

Saranya Chumsri ◽

Mei-Yin Polley ◽

Sarah L. Anderson ◽

Ciara Catherine Maria O'Sullivan ◽

Gerardo Colon-Otero ◽

...

Keyword(s):

Breast Cancer ◽

Phase I ◽

Phase Ii ◽

Triple Negative Breast Cancer ◽

Triple Negative ◽

Response Rate ◽

Single Agent ◽

Objective Response ◽

Locally Advanced ◽

Genomic Study

TPS17 Background: Emerging studies suggest that breast cancer, particularly triple negative breast cancer (TNBC), may be sensitive to immunotherapy. However, the response rate of single agent immune checkpoint blockade agent in TNBC is rather low. Previous genomic study in residual tumor after neoadjuvant chemotherapy showed inverse correlations between MEK activation signature and the amount of tumor infiltrating lymphocytes (TILs) in residual disease samples as well as poor outcome. Preclinical study also showed that the combination of MEK inhibitor and anti-PD-L1 antibody in mouse model can eradicate TNBC tumors. Methods: This is a single arm, Phase I/II trial of Pembrolizumab (P) in combination with Binimetinib (B) in patients with unresectable locally advanced or metastatic TNBC. This trial is currently opened for accrual at Mayo Clinic in Florida and Minnesota. Patients with TNBC defined as ER ≤ 10% and PR ≤ 10% who received ≤ 3 prior lines with measurable disease will be enrolled. The primary objective of the Phase I part is to determine the maximum tolerated dose of B in combination with P and for the Phase II part is objective response rate (ORR) by RECIST criteria. The secondary endpoints include ORR by irRECIST, progression free survival, and overall survival. The total sample size is 15-38 patients with 6-12 patients in Phase I with 2 dose levels and 9-26 patients in Phase II. Simon’s Two-Stage Optimal Design is used to test the null hypothesis that this two-drug combination has an ORR of at most 15% vs. the alternative hypothesis that it has an ORR of at least 35%. Patients will receive single agent B for 2 weeks prior to starting P. A mandatory biopsy will be performed before starting B and an optional biopsy will be performed after 2 weeks of B. Tumor tissue will be evaluated for the amount and phenotypes of TILs, PD-L1 expression, and gene expression analysis using PanCancer Immune Profiling Panel, and PDJ amplification. Peripheral blood will be evaluated for circulating immunoregulatory cells, cytokine profiling, circulating tumor cells (CTCs), as well as p-ERK and PD-L1 expression on CTCs. Clinical trial information: NCT03106415.

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Nivolumab in combination with cabozantinib for metastatic triple-negative breast cancer: a phase II and biomarker study

npj Breast Cancer ◽

10.1038/s41523-021-00287-9 ◽

2021 ◽

Vol 7 (1) ◽

Author(s):

Romualdo Barroso-Sousa ◽

Tanya E. Keenan ◽

Tianyu Li ◽

Nabihah Tayob ◽

Lorenzo Trippa ◽

...

Keyword(s):

Breast Cancer ◽

Phase Ii ◽

Triple Negative Breast Cancer ◽

Primary Endpoint ◽

Triple Negative ◽

Objective Response ◽

Tumor Infiltrating Lymphocytes ◽

Rapid Progression ◽

Immune Gene ◽

Tumor Mutational Burden

AbstractThis single-arm phase II study investigated the efficacy and safety of cabozantinib combined with nivolumab in metastatic triple-negative breast cancer (mTNBC). The primary endpoint was objective response rate (ORR) by RECIST 1.1. Biopsies at baseline and after cycle 1 were analyzed for tumor-infiltrating lymphocytes (TILs), PD-L1, and whole-exome and transcriptome sequencing. Only 1/18 patients achieved a partial response (ORR 6%), and the trial was stopped early. Toxicity led to cabozantinib dose reduction in 50% of patients. One patient had a PD-L1-positive tumor, and three patients had TILs > 10%. The responding patient had a PD-L1-negative tumor with low tumor mutational burden but high TILs and enriched immune gene expression. High pretreatment levels of plasma immunosuppressive cytokines, chemokines, and immune checkpoint molecules were associated with rapid progression. Although this study did not meet its primary endpoint, immunostaining, genomic, and proteomic studies indicated a high degree of tumor immunosuppression in this mTNBC cohort.

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Epigenetic quantification of circulating immune cells in peripheral blood of triple-negative breast cancer patients

Clinical Epigenetics ◽

10.1186/s13148-021-01196-1 ◽

2021 ◽

Vol 13 (1) ◽

Author(s):

Mehdi Manoochehri ◽

Thomas Hielscher ◽

Nasim Borhani ◽

Clarissa Gerhäuser ◽

Olivia Fletcher ◽

...

Keyword(s):

Breast Cancer ◽

T Cells ◽

Triple Negative Breast Cancer ◽

Immune Cells ◽

Triple Negative ◽

Immune Cell ◽

Nk Cell ◽

Cell Type ◽

Cell Ratio ◽

Immune Cell Type

Abstract Background A shift in the proportions of blood immune cells is a hallmark of cancer development. Here, we investigated whether methylation-derived immune cell type ratios and methylation-derived neutrophil-to-lymphocyte ratios (mdNLRs) are associated with triple-negative breast cancer (TNBC). Methods Leukocyte subtype-specific unmethylated/methylated CpG sites were selected, and methylation levels at these sites were used as proxies for immune cell type proportions and mdNLR estimation in 231 TNBC cases and 231 age-matched controls. Data were validated using the Houseman deconvolution method. Additionally, the natural killer (NK) cell ratio was measured in a prospective sample set of 146 TNBC cases and 146 age-matched controls. Results The mdNLRs were higher in TNBC cases compared with controls and associated with TNBC (odds ratio (OR) range (2.66–4.29), all Padj. < 1e−04). A higher neutrophil ratio and lower ratios of NK cells, CD4 + T cells, CD8 + T cells, monocytes, and B cells were associated with TNBC. The strongest association was observed with decreased NK cell ratio (OR range (1.28–1.42), all Padj. < 1e−04). The NK cell ratio was also significantly lower in pre-diagnostic samples of TNBC cases compared with controls (P = 0.019). Conclusion This immunomethylomic study shows that a shift in the ratios/proportions of leukocyte subtypes is associated with TNBC, with decreased NK cell showing the strongest association. These findings improve our knowledge of the role of the immune system in TNBC and point to the possibility of using NK cell level as a non-invasive molecular marker for TNBC risk assessment, early detection, and prevention.

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Fractal dimension, occupancy and hotspot analyses of B cell spatial distribution predict clinical outcome in breast cancer

10.1101/678607 ◽

2019 ◽

Author(s):

Juliana C. Wortman ◽

Ting-Fang He ◽

Shawn Solomon ◽

Robert Z. Zhang ◽

Anthony Rosario ◽

...

Keyword(s):

Breast Cancer ◽

T Cells ◽

Spatial Distribution ◽

B Cells ◽

Clinical Outcome ◽

Immune Cells ◽

Triple Negative ◽

Immune Cell ◽

Statistical Techniques ◽

Killer T Cells

AbstractWhile the density of tumor-infiltrating lymphocytes (TILs) is now well known to correlate with clinical outcome, the clinical significance of spatial distribution of TILs is not well characterized. We have developed novel statistical techniques (including fractal dimension differences, a hotspot analysis, a box counting method that we call ‘occupancy’ and a way to normalize cell density that we call ‘thinning’) to analyze the spatial distribution (at different length scales) of various types of TILs in triple negative breast tumors. Consistent with prior reports, the density of CD20+ B cells within tumors is not correlated with clinical outcome. However, we found that their spatial distribution differs significantly between good clinical outcome (no recurrence within at least 5 years of diagnosis) and poor clinical outcome (recurrence with 3 years of diagnosis). Furthermore, CD20+ B cells are more spatially dispersed in good outcome tumors and are more likely to infiltrate into cancer cell islands. Lastly, we found significant correlation between the spatial distributions of CD20+ B cells and CD8+ (cytotoxic) T cells (as well as CD3+ T cells), regardless of outcome. These results highlight the significance of the spatial distribution of TILs, especially B cells, within tumors.Significance StatementImmune cells can fight cancer. For example, a patient has a good prognosis when a high density of killer T cells, a type of immune cell that can kill cancer cells, infiltrates into a tumor. However, there is no clear association between prognosis and the density of B cells, another type of immune cell, in a tumor. We developed several statistical techniques to go beyond cell density and look at the spatial distribution, i.e., the pattern or arrangement of immune cells, in tumors that have been removed from patients with triple negative breast cancer. We find that B cells and killer T cells tend to be more spread out in the tumors of patients whose cancer did not recur.

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Epigenetic Quantification of Circulating Immune Cells in Peripheral Blood of Triple-negative Breast Cancer Patients

10.21203/rs.3.rs-508197/v2 ◽

2021 ◽

Author(s):

Mehdi Manoochehri ◽

Thomas Hielscher ◽

Nasim Borhani ◽

Clarissa Gerhäuser ◽

Olivia Fletcher ◽

...

Keyword(s):

Breast Cancer ◽

T Cells ◽

Triple Negative Breast Cancer ◽

Immune Cells ◽

Triple Negative ◽

Immune Cell ◽

Nk Cell ◽

Cell Type ◽

Cell Ratio ◽

Immune Cell Type

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Correlation of the tumor mutational burden with the composition of the immune cell subpopulations in peripheral blood of triple-negative breast cancer patients undergoing neoadjuvant therapy with durvalumab: Results from the prospectively randomized GeparNuevo trial.

Journal of Clinical Oncology ◽

10.1200/jco.2019.37.15_suppl.588 ◽

2019 ◽

Vol 37 (15_suppl) ◽

pp. 588-588

Author(s):

Barbara Seliger ◽

Thomas Karn ◽

Carsten Denkert ◽

Andreas Schneeweiss ◽

Claus Hanusch ◽

...

Keyword(s):

Breast Cancer ◽

T Cells ◽

Neoadjuvant Therapy ◽

Triple Negative Breast Cancer ◽

Triple Negative ◽

Immune Cell ◽

Effector Memory ◽

Cell Counts ◽

Mutational Burden ◽

Tumor Mutational Burden

588 Background: The GeparNuevo trial is a randomized, double-blind, multi-center phase II trial of neoadjuvant therapy in patients with early-stage triple negative breast cancer (TNBC) investigating the role of durvalumab, an anti-PD-L1 antibody, which blocks PD-L1 binding to PD1 and CD80, in addition to standard chemotherapy with nab-Paclitaxel (nab-Pac) followed by Epirubicin plus Cyclophosphamid (EC; Loibl S et al. ASCO 2018). Since the tumor mutational burden (TMB) has been suggested to be associated with a better outcome of patients undergoing immunotherapy and an increased T cell response, we determined whether there exists a link between TMB and immune cell composition, frequency and function in patients of the GeparNuevo trial. Methods: In order to determine possible predictive and / or prognostic biomarkers, tumor biopsies taken at recruitment from 149 patients out of the 174 enrolled patients underwent deep sequencing in order to determine the TMB. In addition, for 120 patients blood samples were taken at recruitment and during different time points of treatment (after durvalumab pre-treatment, after Nab-Pac and at surgery after EC) and evaluated using multicolor flow cytometry by monitoring the absolute cell counts of T cells, B cells and NK cells as well as the frequency, composition and functionality of different immune cell populations. Results: The TMB of the GeparNuevo cohort was in line with published data with a mean of 1.8 mutations/MB (range 0.02 – 7.65), respectively. Preliminary evaluation demonstrated a significant correlation of TMB with blood parameters, in particular with subsets of CD8+ T cells. Interestingly, the data suggest a negative correlation of TMB with the frequency of effector cells while a positive correlation exists with the effector memory cells at recruitment. In depth analyses of a correlation with treatment arm and clinical responses are currently performed. Conclusions: Using this approach we hope to identify biomarkers, which will allow a better selection of TNBC patients undergoing specific immunotherapies. Clinical trial information: NCT02685059.

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Comprehensive genomic and immunophenotypic analysis of CD4 T cell infiltrating human triple-negative breast cancer

Cancer Immunology Immunotherapy ◽

10.1007/s00262-020-02807-1 ◽

2020 ◽

Author(s):

He Zhang ◽

Guohui Qin ◽

Hui Yu ◽

Xu Han ◽

Sha Zhu

Keyword(s):

Breast Cancer ◽

Gene Expression ◽

T Cells ◽

Triple Negative Breast Cancer ◽

Triple Negative ◽

Immune Cell ◽

Differential Expression Analysis ◽

Effector Memory ◽

Activated T Cells ◽

Cell Functions

AbstractThe aim of this study is to investigate the gene expression module of tumor-infiltrating CD4+T cells and its potential roles in modulating immune cell functions in triple-negative breast cancer. Differentially expressed genes were identified by comparison of the expression profile in CD4+T cells isolated from tumor tissues and peripheral blood of TNBC patients respectively. The differential expression analysis was conducted using R, and then the functional and pathway enrichment of the DEGs were analyzed using GSEA, followed by integrated regulatory network construction and genetic analysis of tumor-infiltrating immune cells based on a scientific deconvolution algorithm. As a result, abundant Treg and exhausted lymphocytes were detected, accompanied by largely decreased of effector/memory and cytotoxic T cells. Immune-related gene correlation analysis showed that the extent of follicular helper T cells gene expression signatures were inversely associated with those of CD4+ naive T cells and CD4+ memory resting T cells, but positively correlated with that of CD4+ memory activated T cells. In addition, we found five core genes including IFNG, CTLA4, FAS, CXCR6, and JUN were significantly over expressed in CD4+ TILs which may contribute to exhaustion of lymphocytes and participate in biological processes associated with regulation of chemotaxis. Study provides a comprehensive understanding of the roles of DEGs associated with the chemotactic and exhausted immunophenotypes of CD4+ TILs that are a valuable resource from which future investigation may be carried out to better understand the mechanisms that promote TNBC progression.

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