Immune profiling of pre- and post-treatment breast cancer tissues from the SWOG S0800 neoadjuvant trial

Immune cells in the microenvironment of breast cancer influence, and may partially mediate, response to chemotherapy [1–3]. Pre-operative (neoadjuvant) chemotherapy of newly diagnosed, early stage breast cancer where pre- and post-treatment tissues are available provides an opportunity to study baseline immune parameters that are associated with treatment response and to assess changes in the immune microenvironment caused by treatment. Several studies have reported that the higher the tumor infiltrating lymphocyte (TIL) count, or immune-related gene expression at baseline, the greater the probability of pathologic complete response (pCR) to preoperative chemotherapy in all breast cancer subtypes [3–6]. However, the contribution of various lymphocyte and immune cell sub-types to determining treatment response remains unknown. To date, few studies examined immunological changes in the breast tumor microenvironment after chemotherapy [7, 8]. Better understanding of the immune functions that influence the probability of pathologic complete response (pCR) to preoperative chemotherapy could suggest novel immunotherapeutic strategies to enhance chemotherapy efficacy. Patients with extensive residual invasive cancer in the breast or lymph nodes after preoperative chemotherapy have guarded prognosis, particularly if they have triple negative breast cancer [9]. Better understanding of the immune milieu of residual cancers may also suggest novel therapeutic approaches to improve the prognosis of these patients.

S0800 (NCT00856492) was a randomized, 3-arm, Phase II trial that tested if inclusion of bevacizumab with neoadjuvant chemotherapy could improve pCR rates in HER2-negative, locally advanced, or inflammatory breast cancer (IBC). The three arms of the trial were weekly nab-paclitaxel and bevacizumab followed by dose-dense doxorubicin/cyclophosphamide (ddAC) (Arm A), nab-paclitaxel followed by ddAC, (Arm B), and ddAC followed by nab-paclitaxel (Arm C). Patients were randomly allocated in 2:1:1 ratio to arms A, B and C, respectively. For the primary efficacy analysis, the arms B and C were combined. The trial demonstrated that bevacizumab increased pCR rate from 21 to 36% (p = 0.019) but chemotherapy sequence in the non-bevacizumab arms did not significantly influence efficacy [10]. The main objectives of the current study were to (i) examine the association between pCR and pre-treatment TIL count, PD-L1 protein expression and expression of 750 immune-related genes and (ii) assess changes in these immune parameters in paired pre- and post-treatment tissues.

In this study, we examined associations between baseline immune markers and response to nab-paclitaxel and doxorubicin/cyclophosphamide neoadjuvant chemotherapy with or without bevacizumab and assessed changes in the tumor immune microenvironment after treatment. We confirmed earlier observations that higher baseline TIL counts are significantly associated with higher pCR rate even after adjustment for ER and treatment arm [4, 13]. We also identified several immunological functions that provide additional granularity to this association. The significantly higher baseline expression of granzyme and granulysin in the tumor microenvironment of patients who subsequently experience pCR are consistent with the preclinical models that indicate synergy between cytotoxic T cell activity and the antitumor effect of chemotherapy [2, 3]. The high expression of CCL19 and CCL21 in TIL-rich cancers suggests that these chemokines play important roles in attracting T cells, NK cells and mature dendritic cells into the tumor tissues. This raises the possibility re-activation, or supplementation, of these cytokines in immune-poor cancers may turn “immune cold” cancers into “immune hot” tumors. The high baseline expression STAT4, CD7 and IL7R in cases with pCR also suggest that T helper activity and T and B cell maturation are important for achieving pCR whereas the high expression of IL1RN (Interleukin-1 Receptor Antagonist), that suppresses IL1 signaling, suggests that IL-1 activity confers chemotherapy resistance. We were not able to identify any single gene or immune metagene that was selectively predictive of greater sensitivity to bevacizumab. VEGF expression and angiogenesis signatures, had no statistically significant interaction with bevacizumab effect. However, due to the small samples size, the power of this study to detect such interaction is small.

Perhaps more importantly, we also identified a larger number of stroma-related functions and immune parameters that were negatively associated with pCR in all treatment arms. Baseline expressions of mast (MS4A2, CPA3, HDC, TPSB2) and myeloid inflammatory cell metagenes (CXCL1, CXCL3, CXCL2, CCL20, AREG, FOSL1, PTGS2, IER3, IL6) were higher in patients with RD. This raises the possibility that mast cell inhibitors (e.g. masitinib) or IL6 antagonists (e.g. tocilizumab) might convert some patients from RD to pCR. Inhibition of CXCL1, CXCL3, CXCL2, and CCL20 may also increase chemotherapy efficacy. We also noted high baseline expression of several other potential therapeutic targets including VEGFB, TGFB3, PDGFB/PDGFRB, FGFR1 and IGF1R in cancers that did not achieve pCR. The concurrent administration of the anti-VEGF antibody bevacizumab with chemotherapy has increased pCR rates in this and several other randomized neoadjuvant trials (ARTemis, CALGB-40603, SWOG-S0800, NSABP-B40 GeparQuinto), indicating that inhibition of the VEGF axis increases chemotherapy sensitivity. However, two large adjuvant trials (BEATRICE, ECOG-5103) have not shown increased disease-free or overall survival with the inclusion of bevacizumab [16].

When we compared immune parameters in pre- and post-treatment tissues, we noted an almost universal resolution of the immune and inflammatory response in cases with pCR. We previously reported in a larger number of samples from the same study (124 pre- and 62 post-treatment tissues) that TIL counts generally decrease in post-treatment tissues [8]. These observations suggest that either chemotherapy has a cytotoxic effect on TILs or as the size of the primary tumor decreases in response to therapy, the immunogenic target decreases and the corresponding anti-tumor immune reaction also winds down. The greatest decrease in TILs between matched pre−/post-treatment samples is observed in cases with pCR that supports the hypothesis that after complete eradication of the cancer from the breast the immune response also resolves. Indeed, among RD cases, there was no significant difference in pre- and post-treatment TIL counts. However, most immune cell metagene expressions decreased significantly even among cases with RD, except the NK cell metagene that remained the same, and the mast cell metagene that has increased, in post-treatment tissues. The few genes that showed increased expression in residual tissues after pCR included cellular stress and hypoxia induced genes suggesting ongoing tissue repair. In cases with RD, we observed increased expression of a larger number of genes in the residual tissues including several of the same stress- and hypoxia-induced genes (DUSP1, EGR1) as in pCR. The Dual Specificity Phosphatase 1 (DUSP1), is of interest since in some solid tumors it can induce resistance to both chemotherapy and radiotherapy in vitro and in vivo [17]. In addition, we also observed increased expression of IL6 and CXCL2 in RD tissues. Both cytokines were also associated with RD at baseline and represent attractive immuno-oncology targets to enhance chemotherapy efficacy. Several studies implicated high levels of IL6 in poor prognosis and lesser treatment response in breast cancer [18]. The chemokine ligands CXCL-1 and -2 were also shown to promote breast cancer metastasis through myeloid cell recruitment and blocking CXCL-1 and -2 signaling improves chemotherapy efficacy in experimental models [19].

There are a few limitations of this study. Our small sample size prevented us from adequately powered analysis by ER groups, which possess different immunologic characteristics and chemotherapy sensitivities. Several other potentially important subset analyses that we have performed have limited power. We observed no significant difference in survival among residual disease cases that showed increase in TIL count from baseline compared to those that had a decline, we also failed to detect significant association between baseline PD-L1 expression and pCR, as we reported earlier in larger cohorts [5, 8]; these findings most likely reflect underpowered analyses. We also recognize that our pre- and post-treatment sample comparisons may be influenced by unavoidable sampling bias and treatment-related shifts in tumor cellularity. All baseline tissues were from core needle biopsies and all post-treatment samples were from surgically resected tissues and post-treatment samples generally have lower tumor cellularity even in cases with residual disease.

Our analysis suggests that CCL19, CCL21 and IL7 signaling play an important role in attracting and activating immune cells in the breast cancer microenvironment and might help convert immune cold cancers to immune hot. The presence of activated cytotoxic T cells with granzyme and granulysin expression are important for achieving pCR with chemotherapy. The expression of CXCL1, CXCL3, CXCL2, CCL20, and IL6 are enriched in residual cancer and are associated with lesser response the chemotherapy when highly expressed at baseline. These molecules represent potential novel targets to increase pCR rates and improve outcome in patients with residual cancer after chemotherapy.