Elizabeth Mittendorf, left, oversees work in her lab

The excerpt below is from the 2022 Department of Surgery Annual Report. The full report can be read here.

When we think of cancer research and immunotherapy, surgeons don’t immediately come to mind. At the Brigham, however, surgeons are taking a leading role in immuno-oncology research, led by Elizabeth Mittendorf, MD, PhD, director of the Breast Immuno-Oncology Program and co-director of the Breast Cancer Research Program at Dana-Farber Brigham Cancer Center and Ravindra Uppaluri, MD, PhD, chief of the Division of Otolaryngology-Head and Neck Surgery in the Department of Surgery and chief of Head and Neck Surgical Oncology at Dana-Farber Brigham.

“There is a real opportunity for surgeons to be involved in research and to expand research in immuno-oncology, especially in the preoperative setting,” Mittendorf says.

“As surgeons, we have the opportunity to access biospecimens, such as tissue and blood, from patients before, during and after treatment,” Mittendorf explains. “We are uniquely positioned to contribute to cancer research.”

Dozens of immunotherapies are approved for use in the recurrent and metastatic settings, and there is interest in taking this strategy to earlier disease stages. The natural timing, of course, is before surgery. Preclinical studies have shown that stronger and broader immune responses can be generated if immunotherapy is administered while the tumor and/or draining lymph nodes are intact.

At this time, neoadjuvant or preoperative immunotherapy is approved for use in combination with chemotherapy in triple-negative breast cancer and non-small cell lung cancer. The hope is that this approach of harnessing the immune system to fight cancer will also be beneficial in other types of cancer. Analysis of biospecimens collected during surgery are essential for understanding the effects of immunotherapy at the tissue and cellular levels.

“Immunotherapy may be even more effective in the preoperative setting because patients haven’t had a lot of prior therapies that deplete the immune system,” Mittendorf explains.

“By bringing immunotherapy to patients earlier in their disease, we’re trying to advance clinical and functional outcomes, not just surgical outcomes,” Uppaluri says. “There is a subset of newly diagnosed patients who respond amazingly well to immunotherapy and may therefore need less adjuvant radiation or chemotherapy and maybe even reduced extent of surgery.”

“As surgeons this is a big change. Our goal has always been to get a good outcome from surgery,” Uppaluri says. “Now we are enrolling patients in clinical trials to study tumors before and after immunotherapy and gain insights into biological changes in the tumor caused by the drugs.”

“This has been fairly common in breast cancer,” he explains, “but for head and neck cancer, preoperative immunotherapy represents thinking outside the box.”

Immunotherapy Targets

“The vast majority of success in cancer immunotherapy has been in targeting the PD-1 and PD-L1 pathway,” Mittendorf says. “Known as checkpoint inhibitors, PD-1 and PD-L1 put the brakes on T cells.”

When the immune system is stimulated, it sends out “go” signals telling immune cells to recognize and attack the invader and, shortly thereafter, it sends out “stop” signals that are important for keeping the immune system in check. When fighting cancer, though, you don’t want the immune system to stop attacking cancer cells. PD‐1 is considered a checkpoint protein because it enables the immune system to control the action of T cells by either turning activation signals up or down. The current checkpoint inhibitors block checkpoint proteins such as PD‐1 on T cells or PD‐L1 on tumor cells, allowing immune system eradication of cancers.

“This is just the tip of the iceberg,” Mittendorf says. “It’s not all about the T cells. Other cells in the tumor microenvironment, such as B cells, monocytes, macrophages and natural killer cells impact how T cells work.” The objective now is to figure out the additional checkpoint molecules and other cells that play a role in immune system responses to cancer cells.

Ravindra Uppaluri (center) and members of his lab

Over the last two years, Uppaluri and his team have reported promising results from Phase 2 trials of neoadjuvant checkpoint inhibitor therapies in locally advanced squamous-cell carcinomas of the head and neck or oral cavity. Based on their findings, Phase 3 trials are under way to confirm these findings, including the KEYNOTE-689 trial of adjuvant and neoadjuvant pembrolizumab combined with standard of care in patients with resectable, locally advanced head and neck squamous cell carcinoma. Positive results from the Phase 3 trials have the potential to change the way head and neck cancers are treated, explains Uppaluri who serves on the steering committee for KEYNOTE-689.

Although checkpoint inhibitor therapy clearly works for some cancers, others seem to be resistant to this form of immunotherapy. With funding from the National Institutes of Health, Uppaluri’s team has developed mouse models in head and neck cancer that are widely used to study immunotherapy response and resistance. With these models, it is possible to examine neoadjuvant targets of tumor cells as they are seen by immune cells, thus helping to understand how those targets work.

Beyond T Cells

The majority of approved cancer immunotherapy drugs target T cells, the immune system cells that have memory and are able to recognize and remember tumor cells to fight them when they grow.

“What we know now after 20 years of using these therapies clinically, is that not all cancer types respond to T cell therapies,” explains Jennifer Guerriero, PhD, lead investigator in the Division of Breast Surgery. “This has opened the door to thinking about other immune cells in tumors that we might target.”

“There is now growing interest in how myeloid cells, such as macrophages, impact response to immunotherapy,” Mittendorf says. Macrophages are in every tissue and organ and are the most abundant immune cells in tumors. They are the first line of defense against bacteria and pathogens, but they also help with tumor angiogenesis by travelling to tumors and providing factors to help tumors grow.

“Macrophages are very complex cells and there is a lot we need to do to better understand these cells,”  Guerriero explains. “We think macrophages may be one of the reasons T cell therapies don’t not work in some cancers because they interact with T cells and may be mitigating T cell activity.”

Guerriero’s laboratory recently completed RNA sequencing to look at the genetic makeup of each cell. In doing so, they discovered 18 different types of macrophages in hormone receptor-positive breast cancer. Their goal now is to determine what each type of macrophage is doing in each patient’s tumor and how to target it with therapies.

“There is lots of optimism in trying to target macrophages because they are so abundant,” Guerriero says. “Our group, in collaboration with Dr. Mittendorf’s team, is using biospecimens collected during surgery to better understand the different cell populations.” Guerriero’s team is using immunoplex analysis to look at 20 to 30 markers in each cell to identify their locations and spatial relationships in both mouse models and surgical biospecimens.

Guerriero’s team is also using cyclic immunofluorescence, a technique developed in the laboratory of Peter Sorger at Harvard Medical School, which allows the visualization of up to 60 different markers of immune or tumor cells on a single tissue section. In triple-negative breast cancer tumors, they saw that tumor cells (white), macrophages (green) and T cells (red) are in close proximity in the tumor (figure). It will be important to better understand how this spatial relationship influences tumor growth and response to therapy.

Overcoming Resistance to Immunotherapy

“Understanding why some patients don’t respond has been the holy grail in immunotherapy,” Uppaluri says.

A collaborative study with medical oncologist Catherine Wu, MD, at Dana-Farber Cancer Institute (DFCI) is using single-cell RNA sequencing technologies to look at patient samples from clinical trials collected before and after patients receive therapy. The project may yield insights into which subsets of cells are targeted by therapy, what changes over time and what mechanisms may lead to non-responsiveness to therapy.

Two recent finding from Guerriero’s team show the importance of modulating macrophages to help immunotherapy succeed. They found that macrophages treated with a PARP inhibitor inhibited T-cell function and activation, which limited tumor response to PARP inhibitor therapy. This work contributed to the design of a clinical trial evaluating PARP inhibitors plus immune checkpoint inhibitors to further activate T cells as part of the Dana-Farber Breast Program SPORE Award. In another study, they identified a novel strategy to overcome PARP inhibitor resistance through pharmacological targeting of macrophages. This work revealed a previously unknown mechanism of PARP inhibitor resistance and laid the groundwork for future clinical trials to enhance PARP inhibitors plus immune checkpoint blockade by targeting macrophages.

Uppaluri, along with DFCI’s David Barbie, MD, and Robert Haddad, MD, are the principal investigators on a $4.3 million U01 Cancer Moonshot grant from the National Cancer Institute (NCI) and the National Institute of Dental and Craniofacial Research (NIDCR) to understand why many patients with head and neck cancers display resistance to immunotherapy drugs, with the goal of improving treatment response rates for this population.

Sophisticated Immune Cell Analysis

Mittendorf’s team is collaborating with researchers at DFCI, including breast medical oncologist Sara Tolaney, MD, MPH, on sophisticated immune cell analyses to identify biomarkers linked to immune checkpoint inhibitor therapy benefit. In a study of women with metastatic triple-negative breast cancer treated with pembrolizumab and chemotherapy, they identified the following potential biomarkers of response to immune checkpoint inhibitor therapy: high tumor mutational burden; presence of CD8+, CD4 memory T cells, follicular helper T cells and M1 macrophages; and inflammatory gene expression pathways.

The team uses multidimensional molecular profiling techniques to study immune cell phenotypes and specific cell functions in tissue, blood and stool specimens obtained before treatment, after two cycles of treatment and before surgery. Their goal is to determine which patients will benefit from immune checkpoint inhibitor therapy without experiencing toxicities (such as thyroid toxicity or damage to the adrenal glands) and identify associations between immune cell phenotypes and toxicities in the tumor microenvironment. The team is currently working on these analyses for patients treated with neoadjuvant immunotherapy with a $1.9M grant from the Breast Cancer Research Foundation.

The Benefit for Patients

Through multidisciplinary collaborations, the surgeons, medical oncologists, radiation oncologists and researchers at Dana-Farber Brigham Cancer Center continue to explore ways to combine immunotherapy with chemotherapy, radiation and surgery to enhance patients’ response to treatment.

At Dana-Farber Brigham, patients can see all the providers involved in their cancer care in one day. “This seamless interaction among colleagues and patients works very well,” Uppaluri says.

“It is incredible to be able to work so closely with clinical colleagues and surgeons to think about these problems and come up with solutions that will actually benefit patients,” Guerriero says.

“Stimulating the immune system creates adaptive immunity that can continue for weeks, months, years and potentially for life,” Mittendorf says. “Immunotherapy may allow us to one day say that we have cured these patients.”

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