Systemic chemokine-modulatory regimen combined with neoadjuvant chemotherapy in patients with triple-negative breast cancer

Abstract

Background: Higher cytotoxic T lymphocyte (CTL) numbers in the tumor microenvironment (TME) predict pathologic complete response (pCR) to neoadjuvant chemotherapy (NAC) and positive long-term outcomes in triple-negative breast cancer (TNBC). pCR to NAC is achieved only in 30-40% of patients. The combination of NAC with pembrolizumab increases the pCR rate but at the cost of immune-related adverse events (irAEs). Based on these considerations, we tested if systemic infusion of the chemokine modulatory regimen (CKM; selective toll-like receptor 3 (TLR3) agonist rintatolimod, interferon (IFN)-α2b, and cyclooxygenase-2 (COX-2) inhibitor celecoxib) regimen can be safely combined with NAC to enhance intratumoral CTL numbers and NAC effectiveness.

Methods: Phase I study NCT04081389 evaluated nine patients with early-stage TNBC who received 3 weeks of paclitaxel with CKM (dose-escalation of IFN-α2b), followed by 9 weeks of paclitaxel alone, dose-dense doxorubicin and cyclophosphamide, and surgery. Primary and secondary endpoints were safety and clinical efficacy, respectively.

Results: The combination treatment was well-tolerated with no dose-limiting toxicities or irAEs. 5/9 patients achieved pCR and one patient had microinvasive disease (ypTmic). We observed elevated IFN signature and uniform decreases in CTL numbers (average 8.3-fold) in the blood of all treated patients. This was accompanied by reciprocal uniform increases in CD8β (overall 5.9-fold), CD8α/FoxP3 (2.11-fold), and CCL5 (4.73-fold) transcripts in TME, particularly pronounced in patients with pCR. Multiplex immunohistochemistry revealed selectively increased numbers of CTL (but not regulatory T cells) in both the epithelial and stromal tumor compartments and early decreases in the numbers of αSMA⁺ vascular/stromal cells in the tumors of all pCR patients.

Conclusions: Combined paclitaxel/CKM regimen was safe, with desirable TME changes and preliminary indications of promising pCR+ypTmic of 66%, comparable to the combination of NAC with pembrolizumab.

Keywords: Breast Cancer; Chemotherapy; Immunotherapy; Neoadjuvant; Tumor microenvironment - TME.

Figure 1. Study design. (A) Study schema. Systemic chemokine modulation (CKM; 30 min-long IFN-α2b infusion followed by 2.5-hour-long infusion of rintatolimod) was given as nine daily infusions (3 days per week) over 3 weeks (days 0, 1, 2, days 7, 8, 9, and days 14, 15, 16) along with weekly paclitaxel 80 mg/m². CKM consisted of intravenous rintatolimod (200 mg) and oral celecoxib (two doses of 200 mg)±IFN-⍺2b at dose-escalation from 0 (no IFN-⍺2b; dose level 1) through 5 MU/m² (dose level 2) and 10 MU/m² (dose level 3) to 20 MU/m² (dose level 4). Stars represent the timing of tumor biopsies performed before and after the CKM at the higher dose levels. (B) Consolidated Standards of Reporting Trials flow diagram. This depicts screening, enrollment and follow-up of participants in the trial. The trial enrolled nine patients, eight of whom were evaluable for the primary endpoint of safety. IFN, interferon.

Figure 2. Radiologic and pathologic responses post neoadjuvant treatment. Breast MRI responses (left) after completion of neoadjuvant treatment were measured by RECIST V.1.1 and irRECIST for the nine patients on dose levels (DL) 1–4. Waterfall plot shows the % changes in tumor size from baseline to pre-surgery. The clinical stage of the tumors is depicted using AJCC staging. The pathological stage (right) is reported as RCB-0, RCB-I and RCB-II. As of the data cut-off of February 16, 2024, two patients on follow-up progressed and developed metastatic disease and died. The table lists the doses of celecoxib, IFN-α2b, rintatolimod at each DL and occurrences of grade 3 or higher treatment-related adverse events (TRAEs), attributed to paclitaxel, CKM or doxorubicin and cyclophosphamide (AC). Abbreviations: AC, doxorubicin (Adriamycin) and cyclophosphamide; AJCC, American Joint Committee on Cancer; CKM, chemokine modulatory regimen (rintatolimod, IFN-α2b, celecoxib); IFN, interferon; irRECIST, immune related Response Evaluation Criteria in Solid Tumours; RCB, residual cancer burden; RECIST, Response Evaluation Criteria in Solid Tumors.

Figure 3. Decreased CD8⁺T cell counts in the blood of patients directly after CKM with paclitaxel treatment. Acute changes in the immune cell subset composition of peripheral blood were measured at the end (same day) of treatment with CKM and paclitaxel. (A) Multiparameter flow cytometry analysis of circulating lymphocytes (N=8) for patients (PTS) 2–9. The blue lines represent changes in blood in patients who attained a pCR and the red lines represent blood changes in patients with non-pCR. A statistically significant decrease in CD8⁺ T cells and GrB⁺ CD8⁺ T cells was observed. (B) Expression of cytotoxic T lymphocyte markers (CD8α and CD8β) and their effector status (GrB) was measured using RT-PCR for PTS 3–9 (N=7). Data is expressed as ratios of the individual markers to the housekeeping gene, HPRT1. Decreases in both CD8α and CDβ transcripts are observed with no change in GrB transcript measured by RT-PCR (N=7). (C) Short-term changes in the immune signature in peripheral blood mononuclear cells induced by CKM and paclitaxel. Note the changes in chemokines, chemokine receptors, IFN-inducible and IFN-regulatory genes which are upregulated post-treatment (N=7) for PTS 3–9 (>1.2-fold change, FDR<0.1). (D) Combination of CKM and paclitaxel downregulates expression of the Wnt family member 7A, granzyme M, granzyme K, CXCL8, CXCL2, CXCR3, CD8A and CD8B (N=7) for PTS 3–9 (>1.2-fold change, FDR<0.1). (E) xCell immune cell average enrichment scores for the various immune cell subtypes across the pretreatment and same day post-treatment points for PTS 3–9 in the pCR and non-pCR groups. The left panel legend shows the cell subtypes in the order they appear in each stacked bar from left to right. The right panel shows post-treatment decreases in the signatures of the CD4⁺ and CD8⁺ T cells, and a concomitant increase in B cells and macrophage signatures, consistent with their preferential retention within the circulation. CKM, chemokine modulatory regimen; FDR; false discovery rate; IFN, interferon; pCR, pathological complete response RT-qPCR, real-time quantitative polymerase chain reaction.

Figure 4. Intratumoral increases in cytotoxic T lymphocyte markers after systemic CKM and paclitaxel. (A) Intratumoral levels of CD8⍺, CD8β and chemokine expression (normalized for HPRT1) were measured using RT-PCR in tumor biopsies obtained at baseline and at 3 weeks of CKM/paclitaxel regimen (see figure 1 for study schema) for patients on the higher CKM dose levels (PTS 5–9). The blue lines represent patients who attained a pCR (N=4) and the red line represents the non-pCR patient (N=1; dose level 4). Statistically significant increases in CD8β, CD8α/FoxP3, CCL5 and CXCL12 mRNA were observed. (B) Gene Set Enrichment Analysis (GSEA) expression level changes (normalized enrichment scores (NES)) for PTS 5–9 identified multiple immune signaling groups which increased significantly post CKM/paclitaxel, while proliferation gene families were significantly decreased (FDR<0.05). CKM, chemokine modulatory regimen; FDR; false discovery rate; pCR, pathological complete response; PTS, patients; RECIST, Response Evaluation Criteria in Solid Tumors.

Figure 5. CKM and paclitaxel treatment decreases stromal vascular markers while increasing cytotoxic T lymphocytes in the tumors. MICSSS was performed for high-dimensional tissue analysis on tumors at baseline and at 3 weeks at the completion of CKM and paclitaxel. The whole tumor area (excluding normal, necrosis or fibrosis), tumor nests (epithelial) and stromal elements were analyzed. (A) Pseudo-immunofluorescence images from MICSSS used to visualize each marker, are shown for a representative patient with pCR (PT 5) and non-pCR (PT 8), before and after the CKM/paclitaxel. Note that the patient with pCR, but not the non-pCR patient, showed post-treatment CD3 and CD8 infiltration, and decreases in the PanCK-positive and α-smooth muscle actin (α-SMA)-positive cells. Each ROI) is 500 μm × 400 μm. (B) The blue lines represent patients who attained a pCR (N=4) and the red line represents the non-pCR patient (N=1; dose level 4). Treatment with CKM and paclitaxel induces a trend towards closer apposition of CD8⁺T cells to the nearest PanCK⁺ cancer cells in patients with pCR (patients 5, 6, 7, 9) but not in the single patient with non-pCR (PT 8). (C) The blue lines represent patients who attained a pCR (N=4) and the red line represents the non-pCR patient (N=1). There were consistent post-treatment increases in total T cells, CD8⁺ T cells, and CD8⁻ T cells (identified as CD4⁺ T cells) but no changes in regulatory T cells (Tregs) in the epithelial and stromal/vascular areas of the tumors and associated decreases in the prevalence of the cells expressing αSMA, selectively affecting patients who subsequently attained pCR. CKM, chemokine modulation; Multiplexed Immunohistochemical Consecutive Staining on Single Slide, MICSSS; NK, natural killer; pCR, pathological complete response; PT, patient; ROI, region of interest.