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Breast Cancer Immunotherapy Response Markers Emerge From Single-Cell Sequencing Study

NEW YORK – Researchers have used single-cell transcriptome, T-cell receptor, and proteome profiling in breast cancer patients to identify biomarkers that might predict response to neoadjuvant treatment with immune checkpoint inhibitors.  

In a study published in Nature Medicine on Thursday, the scientists, led by a team from Katholieke Universiteit Leuven (KU Leuven) and the Flanders Institute for Biotechnology (VIB) in Belgium, performed single-cell RNA sequencing and single-cell TCR sequencing on samples from 40 patients with hormone receptor-positive or triple-negative breast cancer (TNBC). Of these, 29 patients had received neoadjuvant pembrolizumab (Merck's Keytruda), an anti-PD1 antibody, and 11 neoadjuvant chemotherapy before anti-PD1 treatment. The researchers analyzed samples from patients before they got treatment and while they were on treatment. 

The researchers specifically wanted to explore the immune biomarkers that might be at play in influencing breast cancer patients' ability to respond to immune checkpoint inhibitors, because these tumors haven't been well studied in this regard and anti-PD1/PD-L1 treatment is starting to take hold as a neoadjuvant treatment in breast cancer. "Not all breast cancer patients respond to neoadjuvant immune checkpoint blockade," the authors wrote, adding that although tumor infiltrating lymphocyte (TIL) scores and tumor PD-L1 expression have been proposed as possible response-associated markers, "their efficacies as predictive markers are still unclear."

This study included breast cancer patients regardless of estrogen receptor or HER2 status, as well as a dozen TNBC patients.

Of the 29 patients who received just neoadjuvant pembrolizumab, nine showed T-cell expansion. Of those that did not, 11 had a high percentage of T cells pre-treatment, which didn't expand following pembrolizumab treatment, and nine had "cold tumors" with few T cells before treatment. Moreover, "as much as 61 percent of expanded T cells on-treatment had clonotypes already present pre-treatment," the authors wrote.

The researchers further identified several features that differed between the T-cell expansion and non-expansion groups before treatment, which could be used to predict response to anti-PD1 treatment. For example, the T-cell expansion group had higher expression of CTLA4 and TOX2, along with higher expression of effector and cytotoxic activity-related genes, such as PRF1, GZMB, and IFNG. Patients who did not have T-cell expansion, on the other hand, had decreased expression of RUNX3 and its cofactor CBFB.

They also found that the expression of PDCD1 and CTLA4, along with CD4-positive T-cell activity pre-treatment, were most predictive of T-cell expansion. In non-expanders, some naïve T-cell markers, like CCR7 and LEF1, were higher pre-treatment while immune-checkpoint, tumor-reactive, and co-stimulatory markers were reduced.

The authors acknowledged that because their study was conducted in a window-of-opportunity setting before surgery, they could not determine if patients' T-cell expansion correlated with clinical benefit or tumor response. However, in previous research, T-cell expansion has been associated with increased immunotherapy response in "easy-to-biopsy" tumor types, such as lung cancer and melanoma, they wrote. In this study, patients who had T-cell expansion after treatment also saw a decrease in tumor cells, which the authors said suggests that T-cell expansion may be predictive of anti-PD1 treatment benefit.

The researchers also considered the role of known immunotherapy biomarkers, namely PD-L1 expression and TILs, and noted that PD-L1 expression was only "modestly" associated with T-cell expansion, while TILs could not reliably predict it. Meanwhile, "PD1 and the relative abundance of CD8-positive or CD4-positive T cells emerged as highly predictive markers for T-cell expansion," the authors wrote, adding that gene signatures of immune-checkpoint markers and CD4-positive T-cell activation were also predictive in patients receiving just pembrolizumab, or after chemo in the neoadjuvant setting. Notably, these markers tended to be highly expressed in TNBC patients, compared to hormone receptor-positive patients, which they said makes sense because, to date, most of the immunotherapy benefit has been observed in this breast cancer subtype.

In the US, pembrolizumab is approved in combination with chemotherapy for PD-L1-positive, locally recurrent, unresectable, or metastatic TNBC. However, the US Food and Drug Administration earlier this year decided not to approve pembrolizumab plus chemotherapy as a neoadjuvant and adjuvant treatment for high-risk, early-stage TNBC. While an interim analysis in the Phase III Keynote-522 trial showed that adding pembrolizumab to chemotherapy before surgery could result in a greater percentage of patients experiencing pathologic complete response compared to those receiving just chemo, the FDA wanted more mature outcomes data.

The Belgian study also identified potential therapeutic targets that could supplement treatment with a PD1 inhibitor. The deregulation of RUNX3 found in the non-expansion group suggests, for example, that adding a drug that reactivates the RUNX3 gene could sensitize tumor cells to anti-PD1 treatment, the researchers wrote, and may improve outcomes.

The inhibition of CX3CR1-expressing macrophages or the associated C3 gene could also serve as therapeutic targets, according to the researchers. The CX3CR1-positive macrophages, which can hinder T cells, occurred more frequently in the non-expansion group, suggesting they play a role in immune suppression. If CX3CR1 were inhibited, more T cells may be activated to kill tumor cells, which could also improve treatment response.

Although pembrolizumab is not currently approved for treatment-naïve breast cancer or as a neoadjuvant therapy, Merck has several late-stage clinical trials evaluating pembrolizumab in this setting.

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