Abstract
Background: Somatic alterations in the FGFR pathway (mainly FGFR1, amplified in about 15% of ER+ BC) have been implicated in resistance to endocrine therapy (ET), and more recently, to ...CDK4/6i as well. Based on our preclinical data showing that the FGFR pan-inhibitor erdafitinib when added to fulvestrant/palbociclib resulted in marked PDX regressions, we initiated a phase Ib trial combining erdafitinib with fulvestrant/palbociclib in patients with ER+/HER2-/FGFR-amplified MBC (NCT03238196) to determine safety, tolerability and anti-tumor activity of this combination. Methods: Patients with evaluable ER+/HER2- MBC with FGFR1-4 amplification (detected on tumor next generation sequencing or plasma ctDNA) exposed to at least one ET regimen (but no more than 2 lines of chemotherapy) in the metastatic setting, were treated with fulvestrant, palbociclib (standard of care dosing/ schedule) and oral erdafitinib, tested in 4 doses ranging from 4 mg to 8 mg daily. Once MTD reached, we planned to enroll 20 patients in the expansion portion of the trial. Tumor blocks were collected for FGFR1 FISH amplification analysis, and plasma ctDNA was collected at baseline, 4 weeks and at treatment discontinuation. Tumor assessments were performed every 8 weeks. Results: Since August 2017, 26 eligible patients with evaluable ER+/HER2-/FGFR-amplified MBC were enrolled across 4 institutions. Patient characteristics are summarized in Table 1. Main grade 1 and 2 adverse events (AE) were consistent with on-target toxicities of erdafitinib and/or palbociclib: mucositis (67%), hyperphosphatemia (61%), dysgeusia (52%), diarrhea (48%), fatigue (48%), neutropenia (47%), hand-foot syndrome (38%), anemia (29%), and onycholysis (14%). Febrile neutropenia occurred in 5% patients, no cases of central serous retinopathy were seen. Serious AE were rare: one grade 4 elevation of transaminases (DLT; attributed to fulvestrant), one grade 3 colitis (attributed to erdafitinib), and one thromboembolic event (attributed to palbociclib). In combination with fulvestrant/ palbociclib, the MTD of erdafitinib was 6 mg. No drug-drug interaction was seen. 8 patients were deemed non-evaluable for anti-tumor effect as treatment discontinuation (mainly due to AE) occurred prior to first tumor assessment. Of the 18 evaluable patients: 7 had disease progression, 8 had stable disease (4 of which discontinued treatment due to AE), 3 have not completed their first tumor assessment, 4 are still on treatment; median PFS was 3 months and CBR at 6 months was 28%. However, higher PFS (6 months) was seen in 6/8 patients with high levels of FGFR1 amplification (FISH FGFR1:CEP8 ratio >5; gene copy number >10) and in both patients with FGFR3 amplification.
Conclusion: To our knowledge, this is the first time an FGFR inhibitor has been tested in combination with ET and CDK4/6i in patients with MBC harboring FGFR alterations. Erdafitinib-related side effects appeared to be on target, leading to treatment discontinuation in several patients despite optimal medical treatment. Clinical activity was seen in heavily pre-treated patients with molecular evidence of high FGFR amplification despite 100% prior exposure to ET and CDK4/6i. Full clinical and correlative work will be presented at the meeting, and a future phase II trial is being planned.
Table 126 / 35 patients accrued13 in escalation, 13 in expansion(ongoing)Median age53 (35 - 75)Race/ ethnicityWhite 22Black 1Asian 2Hispanic 1Median number of lines of treatment in the metastatic setting4 (1 - 5)Prior lines of treatment in the metastatic settingEndocrine therapy 100%Fulvestrant 28%CDK4/6i 100%PI3K pathway inhibitor 80%1 line chemo 65%2 lines chemo 45%FGFR1 amplification23FGFR3 amplification2FGFR4 amplification1
Citation Format: Ingrid A. Mayer, Barbara B. Haley, Vandana G. Abramson, Adam Brufsky, Brent Rexer, Erica Stringer-Reasor, Komal L. Jhaveri, Melinda Sanders, Paula I. Ericsson-Gonzalez, Fei Ye, Carlos L. Arteaga. A phase Ib trial of fulvestrant + CDK4/6 inhibitor (CDK4/6i) palbociclib + pan-FGFR tyrosine kinase inhibitor (TKI) erdafitinib in FGFR-amplified/ ER+/ HER2-negative metastatic breast cancer (MBC) 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 PD1-03.
Abstract
Purpose: Neoadjuvant chemotherapy (NAC), the standard of care for a subset of breast cancer patients, is known to have immunologic effects. With emerging data showing improved response rates ...with anti-PD-1/PD-L1 immunotherapy in combination with chemotherapy, the effects of NAC on systemic and local anti-tumor immunity require further study. Biomarkers of anti-tumor immunity are needed to identify which patients are most likely to respond to immunotherapy. Our previous work has shown that changes in the peripheral blood can be observed over the course of NAC for breast cancer. Peripheral blood biomarkers are attractive because of the relative ease of sampling compared to site of disease. Residual cancer burden (RCB) is a useful surrogate marker of long-term prognosis, as patients who experience a pathologic complete response (pCR) have better outcomes than those with residual disease (RD). Methods: We previously identified an 8 gene signature of cytotoxicity, derived from single cell RNA sequencing of PD-1Hi CD8+ T cells, which are enriched for tumor-reactive T cells. Using a custom NanoString panel, we tested expression of this gene signature in whole blood collected prior to definitive surgery in 88 breast cancer patients (TNBC, n=21; HER2+, n=17; ER+, n= 54; PR+, n=53) across two cohorts (VUMC, n=58; DFCI, n=30), 64 of whom had received NAC (pCR, n=11; RD, n=53). We further investigated peripheral blood gene expression using RNA sequencing (n=58; 34 post-NAC, 24 untreated). Results: In two cohorts of breast cancer patients, expression of the 8 gene signature (FGFBP2 + GNLY + GZMB + GZMH + NKG7 + LAG3 + PDCD1 - HLA-G) was highest in patients with RD who experienced a recurrence within three years compared to those with pCR (p<0.01) or those with the highest RCB (RCB III) compared to those with RCB 0/I/II who did not have a recurrence with three years (p<0.05). RNA sequencing showed higher expression of interferon alpha, interferon gamma, and complement gene sets in patients experiencing a pCR compared to those with RD by gene set enrichment analysis (FDR-corrected q-values < 0.05). Conclusions: Expression of immune-related genes in the peripheral blood may predict response to NAC in breast cancer patients and be a useful biomarker for those who would benefit from additional therapies. These results will be further tested in a large cohort of longitudinal samples from breast cancer patients receiving NAC alone or in combination with pembrolizumab from the I-SPY-2 trial, to determine whether peripheral blood gene signatures can predict response to immunotherapy in breast cancer.
Citation Format: Margaret L Axelrod, Paula I Gonzalez-Ericsson, Xiaopeng Sun, Riley E Bergman, Joshua Donaldson, Sara M Tolaney, Ian E Krop, Ana C Garrido-Castro, Melinda E. Sanders, Ingrid A Mayer, Justin M Balko. Peripheral blood gene signatures predict response to neoadjuvant chemotherapy in 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 PD9-06.
Abstract
Cyclic dinucleotide (CDN) agonists of stimulator of interferon genes (STING) activate innate immunity to increase tumor immunogenicity. However, the efficacy of CDNs is limited by drug ...delivery barriers, including inefficient transport to the cytosol where STING is localized. We recently developed STING-activating nanoparticles (STING-NPs), polymer vesicles designed for enhanced cytosolic delivery of the endogenous CDN ligand for STING, 2’3’ cyclic guanosine monophosphate-adenosine monophosphate (cGAMP). Intratumorally-administered (IT) STING-NPs significantly enhance the therapeutic efficacy of cGAMP and improve response to immune checkpoint inhibition (ICI) in established murine B16 melanoma tumors. However, the immunologic effects of STING-NPs have not been well characterized in tumors or lymphatic tissue.
We utilized Digital Spatial Profiling (DSP) to identify immunologic responses in the injected tumor, a distal tumor, and both tumor draining lymph nodes (TDLN) 48 hours after a single IT injection of STING-NP or three injections of STING-NP co-administered with systemic ICI (anti-PD-1/CTLA-4). DSP analysis permits simultaneous detection of over 30 protein markers in distinct spatial regions of interest (ROI; n=2-4 per tumor/TDLN) or cell types in tissue sections.
After a single STING-NP injection of B16 tumors, B7-H3, an immunosuppressive T cell checkpoint, was significantly induced in tumors compared to PBS or free cGAMP. More strikingly, in T cell-rich regions of the TDLN, STING-NP dramatically increased B7-H3, GZMB, and S100A9 while suppressing VISTA and CD73 expression. After multiple injections of STING-NP, B7-H3, S100A9, CD73, Foxp3, and beta-catenin were substantially upregulated in the injected tumor, with more modest fold changes in distal tumors. Co-treatment with systemic ICI in part abrogated this effect. In contrast, STING-NP-induced changes in GZMB, CD4, and CD8a expression were observed regardless of ICI treatment, and were generally equivalent in both treated and distal tumors, suggesting an abscopal effect. Both the treated and distal TDLN showed striking S100A9 increases which was preferential, but not exclusive, to T cell-rich vs. B cell regions. This effect was exacerbated with the combination of STING-NP and ICI therapy. B7-H3 upregulation in response to STING-NP (regardless of ICI) was exclusive to treated TLDNs vs. distal TDLNs. B7-H3 was expressed in both B and T cell regions, but was nearly two-fold higher in B cell-rich regions. Upregulation of GZMB (5-10 fold over free cGAMP alone) was observed in both treated and distal TDLNs.
Markers of T cell activation were observed after STING-NP treatment, including moderate abscopal effects in synchronous distal tumors. In TDLNs, STING-NP robustly induced S100A9 and B7-H3, which may be immunosuppressive escape mechanisms that could be targeted to enhance responses.
Citation Format: John T. Wilson, Daniel Shae, Paula I. Gonzalez-Ericsson, Violeta Sanchez, JingJing Gong, Yan Liang, Douglas Hinerfeld, Joseph M. Beechem, Justin M. Balko. Digital spatial profiling of molecular responses to nanoparticle STING agonists identify S100A9 and B7-H3 as possible escape mechanisms abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4978.
Abstract
Immune checkpoint inhibitors (ICI) have improved patient overall and progression-free survival in some cancer types with limited success in breast cancer. Clinical trials in triple negative ...breast cancer (TNBC) patients, who harbor extensive tumor-infiltrating lymphocytes within tumor stroma, have demonstrated increased progression-free survival (IMpassion130) and pathologic complete response (KEYNOTE-522). Thus, combinations of ICI and chemotherapy have been FDA-approved for metastatic TNBC. However, the therapeutic benefit of ICI alone is poorly characterized. We sought to model ICI response in vivo to ascertain the immune repertoire responsible for ICI efficacy in breast cancer and identify the therapeutic benefit of ICI alone or in combination with approved chemotherapeutics.
We used an immunocompetent EMT6 orthotopic mammary tumor model to investigate the efficacy of single-agent ICI (anti-PD-L1) or in combination with standard-of-care chemotherapy (paclitaxel or doxorubicin). Analysis of the primary tumor immune landscape was performed by flow cytometry and single-cell RNA sequencing. Peripheral blood from mice was serially sampled by bulk and T-cell receptor (TCR) sequencing to identify systemic genomic alterations and T-cell expansion, respectively.
Single-agent ICI robustly suppressed primary tumor growth (p =0.0046) and extended survival (p<0.0001) beyond the control group. While chemotherapy demonstrated moderate therapeutic efficacy, it did not enhance ICI benefit. Transcriptomic and phenotypic profiling of the tumor microenvironment (TME) revealed increased T cells, dendritic cells, and NK cells in the combination groups versus chemotherapy alone, but this did not translate into improved benefit. Interestingly, despite using a genetically identical tumor model and murine host, ICI induced heterogeneous responses, ranging from complete response to intrinsic resistance. The longitudinal analysis of peripheral blood from heterogeneously responding mice uncovered enriched myeloid signatures and clonal T cell expansion corresponding to ICI resistance and response, respectively.
In conclusion, we identify a heterogeneously ICI-responsive in vivo model that emulates TNBC patient response to combinatorial ICI approaches. We report the efficacy of single-agent ICI in upregulating cytotoxic immune cell infiltration and expansion within the primary tumor, thereby diminishing tumor growth and enhancing survival. We describe host-specific signatures, specifically myeloid cells, that correlate with differential responses to immunotherapy, which models heterogeneous patient response to ICI. Ongoing characterization of matched peripheral blood and TME samples may identify systemic biomarkers and tumor antigen-specific T cell clones to accurately predict ICI response in patients and uncover mechanisms for sensitizing tumors refractory to ICI.
Citation Format: Ann Hanna, Xiaopeng Sun, Paula I. Gonzalez-Ericsson, Violeta Sanchez, Justin M. Balko. Host myeloid response drives anti-PD-L1 resistance in murine models of triple negative breast cancer abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2726.
Abstract
The recent approval of anti-PD-L1 immunotherapy in combination with nAB-paclitaxel for metastatic triple-negative breast cancer (TNBC) highlights the need to understand the role of ...chemotherapy in modulating the tumor-immune microenvironment (TIME). Patients with TNBC are routinely treated with neoadjuvant chemotherapy (NAC). Stromal tumor-infiltrating lymphocytes (sTILs) in the pre-treatment diagnostic biopsy are predictive of pathologic complete response (pCR). In patients with residual disease (RD) at surgery, sTILs confer good prognosis. However, the effect of chemotherapy on sTILs and how it influences the TIME are poorly understood. We examined immune-gene expression patterns before and after NAC in a series of 83 breast tumors, including 44 TNBCs, from patients with RD. sTILs were enumerated by standardized guidelines. Gene expression patterns were tested for association with recurrence-free (RFS) and overall survival (OS). T cell receptor sequencing (TCRseq) was performed on a subset (n=15) of tumors. In 4 patients undergoing NAC, PD-1-high and -negative CD8+ peripheral blood mononuclear cells (PBMCs) were profiled using single-cell RNAseq and multiplexed cytokine secretion assays. Post-NAC sTILs (≥30%) were only predictive of outcome (RFS p=0.019; OS p=0.05) in TNBC patients, but not in non-TNBC patients (RFS p=0.28; OS p=0.78) confirming that the prognostic capacity of sTILs is confined to TNBC. Pre-NAC sTILs were not predictive of outcome in either group, likely due to exclusion of patients experiencing pCR. The change in sTILs during NAC did not prognosticate outcome in TNBC, suggesting that in the post-NAC setting, only the most proximal measurement of sTILs is meaningful. However, these results did suggest that NAC alters the TIME. To examine the interplay among NAC, the TIME, and clinical outcomes, we tested the change in expression of 770 immune-related genes during NAC in univariate cox-proportional hazards models. In non-TNBC, no change in expression of any single gene was associated with RFS or OS at a false-discovery rate (FDR) of 10%. In TNBC, individual changes in 12 genes and 204 genes were identified as associated with RFS and OS, respectively (FDR<10%). Interestingly, in nearly all cases, upregulation of these genes during NAC was associated with improved outcome, with only 1 and 15 genes being associated with poor RFS and OS, respectively. Collapsing genes to functional and cell-type specific signatures gave similar insights: T cell, NK cell, TNF-superfamily, and toll-like receptor signatures were highly prognostic. Surprisingly, NAC did not alter T cell clonality in TNBC. Thus, the immunologic impact of chemotherapy appears to be specific to TNBC and is primarily a beneficial effect but does not appear to appreciably expand the clonality of tumor-infiltrating T cells. Using fresh PD-1HI CD8+ T cells isolated from PBMCs of patients undergoing NAC, we detected a significant increase in cytolytic and inflammatory cytokines secreted in 2 TNBC patients after chemotherapy, but not in 2 non-TNBC patients, which was particularly dramatic in one TNBC patient who experienced a pCR. A further characterization of PD-1HI CD8+ cells by single-cell RNAseq identified a sizeable expansion of cytolytic gene (granulysin, Ksp37, granzyme) expressing cells in the TNBC patient with pCR compared to the TNBC patient with RD. In conclusion, we have characterized the effects of NAC on the TIME. TNBC appears to be uniquely sensitive to the immunologic effects of NAC, and most of these effects are primarily stimulatory, rather than repressive. Finally, these changes can be observed in the PD-1HI CD8+ peripheral T cell compartment and appeared to co-occur with pCR.
Citation Format: Justin M Balko, Mellissa Nixon, Paula I Gonzalez-Ericsson, Mark A Pilkinton, Wyatt J McDonnell, Violeta Sanchez, Susan R Opalenik, Sherene Loi, Brent Rexer, Vandana Abramson, Valerie Jansen, Simon Mallal, Jonathan D Marotti, Kevin Shee, Todd W Miller, Melinda E Sanders, Ingrid A Mayer, Roberto Salgado. Immunologic correlates of long-term outcome in the residual disease of triple-negative breast cancer after neoadjuvant chemotherapy 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 P3-08-15.
We introduce quanTIseq, a method to quantify the fractions of ten immune cell types from bulk RNA-sequencing data. quanTIseq was extensively validated in blood and tumor samples using simulated, flow ...cytometry, and immunohistochemistry data.quanTIseq analysis of 8000 tumor samples revealed that cytotoxic T cell infiltration is more strongly associated with the activation of the CXCR3/CXCL9 axis than with mutational load and that deconvolution-based cell scores have prognostic value in several solid cancers. Finally, we used quanTIseq to show how kinase inhibitors modulate the immune contexture and to reveal immune-cell types that underlie differential patients' responses to checkpoint blockers.Availability: quanTIseq is available at http://icbi.at/quantiseq .
Abstract
Triple-negative breast cancers (TNBCs) are highly heterogeneous and aggressive, with high mortality rates. Although TNBC is typically more responsive to chemotherapy than other breast cancer ...subtypes, many patients develop chemo-resistance. The molecular processes between tumor and stromal cells involved in developing chemo-resistance are under-explored. Here we report studies of paired TNBC patient-derived xenografts (PDX) established before and after chemo-resistance. Despite significant genetic similarities, the chemo-resistant PDX model harbored a rare constitutively-active KRASQ61R mutation which was not present in the chemo-naive PDX. Further analysis demonstrated that the chemo-resistant KRAS-mutant model exhibited altered gene expression changes including increased expression of CXCR2-ligands CXCL1 and CXCL2, which are responsible for recruiting immune cells to tumors. These expression patterns were largely inhibited in vivo by MEK inhibitor (MEKi) treatment. Moreover, in breast cancer cell lines, CXCL1, CXCL2, and granulocyte macrophage-colony stimulating factor (CSF2, stimulates granulocyte and macrophage differentiation from hematopoietic precursor cells, including immunosuppressive myeloid cells) transcripts were also downregulated by MEKi. Notably, chemo-resistant KRAS-mutant tumors harbored increased Gr1+ and Arginase-1+ cells, consistent with recruitment of immunosuppressive M2-like macrophages and/or myeloid-derived suppressor cells (MDSCs), which was inhibited by MEKi. Further experiments demonstrate that CD45+CD11b+Ly6G+ MDSC accumulation in tumors can be inhibited by MEKi treatment alone, or by CXCR2 inhibition, suggesting that the effects of MEK inhibition on MDSC recruitment are CXCL1/2-dependent. Confirming the translational relevance of these findings, in >200 murine and >1000 human breast tumors, Ras/MAPK transcriptional activity correlated with myeloid-recruiting CXCL1/2 expression and negatively with T-cell recruiting chemokines (CXCL9/10/11), even in the absence of activating KRAS mutations. The association with Ras/MAPK activity was also confirmed using immunofluorescence to quantify MHC-II-low myeloid cells in 80 post-chemotherapy TNBC tumors. Importantly, MEKi and chemotherapy combination treatment reversed immunosuppressive cell accumulation and metabolic phenotypes exemplified by altered optical redox ratios (NAD(P)H/FAD) in the chemo-resistant KRAS mutant tumors, resulting in tumor growth suppression in mice. MEKi treatment also reduced redox ratios in 3D cultures of breast cancer cell lines further suggesting that MEK inhibition targets multiple oncogenic processes in breast cancer. These results suggest that Ras/MAPK pathway inhibitors may be effective in some breast cancer patients to reverse Ras/MAPK-driven tumor metabolism and immunosuppression, particularly in the setting of chemo-resistant disease.
Citation Format: Derek A. Franklin, Joe T. Sharick, Paula I. Ericsson-Gonzalez, Violeta Sanchez, Phillip T. Dean, Susan R. Opalenik, Stefano Cairo, Jean-Gabriel Judde, Michael T. Lewis, Jenny C. Chang, Melinda E. Sanders, Rebecca S. Cook, Melissa C. Skala, Jennifer Bordeaux, Jehovana Orozco Bender, Christine Vaupel, Gary Geiss, Douglas Hinerfeld, Justin M. Balko. MEK activation modulates immunosuppressive MDSCs and metabolic phenotypes in TNBC abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1511.
This article has been withdrawn by the authors. We discovered an error after this manuscript was published as a Paper in Press. Specifically, we learned that the structures of glycans presented for ...the PD-L1 peptide were drawn and labeled incorrectly. We wish to withdraw this article and submit a corrected version for review.
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