Detecting tumor responses before they become deadly

Targeted cancer therapies can be stunningly effective at blocking specific, cancer-driving signals. But all too often, tumors develop resistance by switching to alternative signaling pathways to resume their life-threatening drive to multiply and spread.

In a step toward longer lasting treatment, researchers have shown how it’s possible to track the evolution of a person’s cancer quickly and closely enough to detect resistance mechanisms as they arise.

“This is a demonstration that we can learn enough about an individual’s cancer to understand how it’s escaping control, and we can get the data quickly enough that it can be used to help us understand what to do next for the patient,” said Joe Gray, Ph.D., professor emeritus of biomedical engineering in the OHSU School of Medicine. Gray is a senior author of a paper in Cell Reports Medicine describing the research, carried out in the Knight Cancer Institute’s SMMART program with support from the Human Tumor Atlas Network.

Image: Focused ion beam scanning electron microscopy unveils interactions between tumor cells (red) and stromal cells (blue). Long cellular protrusions provide intimate contact between stromal and cancer cells while also tightly linking tumor cells to each other within the tumor nest.Co-author Brett Johnson, Ph.D., said the work was only possible through the combined efforts of an interdisciplinary team of clinicians and scientists. “Researchers analyzing patient samples worked with computational biologists to integrate their results. Close collaboration with oncologists, radiologists, pharmacists, and pathologists allowed us to better understand that data and merge it with the patient’s clinical information.” Johnson is senior manager, research analytics, for the SMMART program.

The study focuses on the case of a woman diagnosed with breast cancer at age 64. She had undergone surgery, radiation therapy and multiple rounds of chemotherapy when scans revealed that the cancer had returned and spread widely in her body. That was when she enrolled in the SMMART program, in which Knight Cancer scientists and clinicians have made it possible to study each person’s cancer in great detail to reveal how tumor cells are responding to treatment.

SMMART looks much deeper than the gene mutations known to drive tumors. It includes analysis of tumor proteins, the microenvironment around cancer cells, immune system interactions, and electron microscope imaging of cancer cell structures. A SMMART team is present during tumor biopsy procedures so that they can begin preservation of the tumor tissue within minutes to preserve features that would otherwise decay.

SMMART team members discuss test results during a clinical tumor board. From left to right: Gordon Mills, M.D., Ph.D., Joe Gray, Ph.D., Christopher Corless, M.D., Ph.D., and Jamie Keck, Ph.D. (OHSU/Kristyna Wentz-Graff)

Well-designed workflows enable the team to perform all of the SMMART measurements and tests using material from a single biopsy. And the analyses are completed quickly. Detailed findings that would normally take months or even years to report are being made available to treating physicians within a few weeks.

The team followed the woman with metastatic breast cancer through four treatment phases over a 3.5-year period. Her physician made management decisions based on clinical information plus input from a multidisciplinary tumor board. Each of the first three treatment phases managed to control tumor growth temporarily, with a new phase of therapy beginning at signs of progression.

The researchers were able to collect tumor samples over the full range of treatment: a primary breast tumor, a liver biopsy taken immediately prior to phase 1, a biopsy of a different liver lesion taken at the end of phase 1, a bone lesion biopsy taken at the end of phase 3, and a biopsy of a third liver lesion taken at the end of phase 4. The researchers used their pipeline of tests and microscope images to generate what they call an omic and multidimensional spatial atlas of the evolving breast cancer.

“The most important point here is that each tool gives you a different perspective on the cancer and, in ensemble, they give you a much better understanding of what’s going on as it evolves,” Gray said. This broad and deep approach can uncover mechanisms of drug resistance that would be missed by limiting analyses to one or a few analytical platforms, he and co-authors assert.

SMMART clinical trials are underway for a variety of cancers, including breast, ovarian, sarcoma, and pancreatic. Patients interested in learning about opportunities can contact Clinical Research Manager Kiara Siex (siex@ohsu.edu).For example, it took a combination of analyses of biopsy 2 to suggest a likely mechanism of resistance to the drug palbociclib, an inhibitor of the enzyme called CDK4/6. Loss of the gene RB1A is the typical driver of resistance to palbociclib, but this gene was not mutated or deleted in biopsy 2. Evidence from protein profiling of key cell cycle regulators revealed that RB1 might have been acted on by the enzyme CDK2, which also inhibits RB1 but is not a target of Palbociclib. The researchers noted that the CDK4/6 inhibitor abemaciclib has a broader spectrum of activity that might make it effective in cases where palbociclib escape occurs via CDK2 activation. And, in fact, abemaciclib given after the period covered by this study showed efficacy.

Comparative analyses of the primary tumor and serial biopsies suggested several mechanisms shaping immune system interactions. The most significant coincided with palbociclib treatment at the time of biopsy 2. The researchers found an increase in macrophages, monocytes and T cells and a decrease in regulatory T cells in biopsy 2 compared with biopsies 1 and 4. This change observed in biopsy 2 may have supported T cell activation, as evidenced by increased expression of the immune checkpoint protein called PD-1. The findings suggest that drugs called checkpoint inhibitors could be used to stop the cancer at this point. And in fact, phase 2 and 3 treatments with the checkpoint inhibitor pembrolizumab were associated with a decrease in the biopsy 1 and 2 lesions. The actual impact of pembrolizumab, however, is uncertain because it was given with other drugs, the researchers point out.

Electron microscope imaging revealed several significant structural changes amid tumor cells that might influence response to treatment. For example, tumors displayed many long thin protrusions reaching into their surrounding environment.

“It looks like they have many functions: communication, sampling the environment, giving cancer cells the force to move,” Gray said. “If you can cut off those functions, it might reduce motility, metastasis, and make the response to targeted drugs more effective.”

Focused ion beam scanning electron microscopy of a metastatic breast cancer cell reveals long, thin protrusions reaching into the surrounding microenvironment, allowing the cell to participate in cell signaling and protein uptake.

Electron microscopy also revealed an abundance of structures, called macropinosomes, formed when cells engulf material from their surroundings. Nutrient scavenging from the intercellular space and nearby dying cells is a known tumor survival mechanism. The researchers note that drugs linked to proteins might be able to convert this survival mechanism into a therapeutic vulnerability.

A high prevalence of densely stained vesicles within tumor cells appear to be lysosomes, which cancer cells can use to trap cancer drugs. Lysosome sequestration has been implicated as a mechanism of resistance to CDK4/6 inhibitors, which may have occurred in the study patient: the researchers recorded a sharp increase in lysosomes during the period between the first and second biopsies. It may be possible to counter this resistance mechanism with drugs such as propranolol, the researchers note.

Altogether, the findings shows that a multitude of deep analyses of tumor responses can be executed routinely and safely. And such combined analyses can reveal potential new mechanisms of resistance and suggest novel therapeutic vulnerabilities.

“We’ve made all of our data and methods available through the Human Tumor Atlas Network,” Johnson said. “Just as we’re building upon the successes of this study in the SMMART Program, our goal is for the research community to also use this as a stepping stone towards better understanding and treating evolving cancers.”

Several of the methods brought to bear in SMMART are too complex to be used widely in the clinical care of cancer patients. But the researchers say that workflows can be simplified and streamlined as the usefulness of an assay platform is established. “Once we understand what the phenomena are that are causing cancers to become resistant, we can come up with much simpler essays to obtain the information,” Gray said. “Our long term goal is to learn enough to look in the blood for those same kinds of events so that we don’t have to do biopsies.”

Further reading:

An omic and multidimensional spatial atlas from serial biopsies of an evolving metastatic breast cancer by Brett E. Johnson,  Allison L. Creason, Jayne M. Stommel, Jessica L. Riesterer, Zahi Mitri, Gordon B. Mills, Joe W. Gray and others, Cell Reports Medicine (Feb. 15, 2022)

The Human Tumor Atlas Network: Charting Tumor Transitions across Space and Time at Single-Cell Resolution by Orit Rozenblatt-Rosen, Aviv Regev, Philipp Oberdoerffer, Emek Demir, Jeremy Goecks, Joe W. Gray, Robert B. West, and Elizabeth H. Williams. Cell (April 16, 2020)

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