Cancer is the second leading cause of death worldwide and the incidence is rapidly increasing. Surgical excision remains the corner stone of treatment. Therefore, the need for implementation of technical improvements is evident. Especially as we are entering an era of endoscopic and robotic surgery.
After surgical tumor excision, the observation of residual tumor tissue remains a major frustration for patients and surgeons. Usually, the residual tumor tissue, or so-called tumor positive margin, is diagnosed a couple days after surgery by the pathologist. This occurrence has major effects on patients’ perspective and mostly necessitates additional treatment pathways. Despite promising technical advances in the past two decades, the amount of tumor positive margins remains high. More precisely, an incidence of 5-30% depending on the tumor type, anatomical location and disease stage. In most solid tumors, tumor positive margins are correlated with a decreased overall- and disease-free survival. Therefore, it is often followed with adjuvant treatment including chemotherapy, radiotherapy or a new surgical intervention.
The cancer burden can be reduced through early detection of cancer. Fluorescence-guided surgery, a worldwide expanding optical imaging technique, increases tumor visualization where it matters the most: at the surgical theatre, with the ultimate goal to increase the detection of residual tumor and decrease the amount of tumor positive margins in a variety of tumors. In this case study, we will guide you through a by us conducted Phase I study of a novel tumor targeting method developed by one of our clients. Their imaging agent has the potential to greatly improve surgical visualization using a tumor-generic fluorescent tracer.
Why a tumor-generic fluorescent tracer?
Tumor specific fluorescence-guided imaging uses a variety of tumor targeting techniques. For example, fluorophore-labeled small molecules, antibodies, nanobodies, peptides or nanoparticles against cell surface receptors. Despite promising results, these strategies often lack broad tumor applicability due to a great diversity of oncogenotypes and phenotypes. Therefore, a more generic assessment is needed to allow for easy implementation of fluorescence-guided imaging. For effective tumor proliferation, tumors convert glucose into lactate, resulting in an acidic extracellular environment. In turn, this makes tumor acidosis a generic target for most solid tumors.
A tunable pH-sensitive polymer.
Our client, an American biotech organization, developed a series of tunable pH-sensitive polymers. Those polymers are labeled with indocyanine green (ICG), a well-known and widely used FDA-approved fluorophore which can be visualized with a variety of commercially available fluorescence cameras. In a normal (e.g., benign) environment, the ICG is quenched. In other words, a decreased fluorescence intensity of the tracer. When the polymers reach an acidic environment, for example in tumor tissue, the polymers dissociate. This process unquenches the ICG, and thus, tumor-tissue specific fluorescence visualization is visible. The dissociation of the polymers is irreversible. This prevents deactivation of the fluorescence signal, allowing for a long period of stable tumor-specific imaging.
Moreover, this so-called nanoscale cooperativity overcomes metabolic and phenotypic variability between different patients and tumors. This is a broad advantage over for example antibody-targeted imaging. Namely, in antibody-targeted imaging a patient is in need of an overexpression of a certain tumor receptor, which differs in between patients and in between tumor types. For example, at the UMCG, they use bevacizumab-800CW (target VEGF-A) and cetuximab-800CW (target EGFR), which have shown promising results in tumor targeting. [1–3] However, only in a genuinely selected array of tumor types like breast cancer, sarcomas and head-and neck cancer.
Phase I study workflow & data gathering.
Together with TRACER our client performed a Phase I clinical trial study on the clinical use of pH probes in tumor targeted therapy, as published in Nature Communications in June 2020.  The study evaluated the safety, pharmacokinetics and feasibility of the pH-activated imaging agent in image-guided surgery, occult tumor detection and visualizing tumor borders in four different cancer types. Namely, head and neck squamous cell carcinoma, breast cancer, esophageal cancer and colorectal cancer. After extensive medical screening, patients received the pH-sensitive probe intravenously 24 hours prior to surgery (Figure 1). During surgery, fluorescence-guided imaging was performed using a commercially available open fluorescence camera. Directly after surgery, fluorescence imaging was performed on the excised specimen. Fluorescence activation on the excision margin was suspected for a potential tumor positive margin. During histopathological analysis, fluorescence imaging was performed using a closed field imaging device. This allowed for adequate discrimination of fluorescence signals between tumor tissue and healthy/benign tissue. All imaging results were correlated final histopathology. The patient inclusion was performed within nine months.
Figure 1 – Workflow
Results: pH probes in tumor targeted therapy can be clinically relevant.
The study indicated that the low pH resulting from tumor acidosis can be exploited as a generic biomarker for cancer in patients with at least four different solid tumor types. Due to the binary off/on mechanism of this pH-activated tracer, a sharp delineation between tumor tissue and benign tissue was observed, both in vivo and ex vivo. In thirty subjects, intraoperative detection of all tumor-positive surgical margins (9 out of 9) was observed. This showed the potential for surgical improvement in the future. Moreover, the detection of occult diseases like ductal carcinoma in situ, a satellite metastasis and peritoneal metastasis show the broad applicability of pH-activated imaging during surgery.
A potential drawback could be the activation of the pH-activated tracer by other mechanisms associated with acidosis, like for example inflammatory tissue. Peri-tumoral inflammation is part of the defense mechanism of the innate immune system to the invading tumor cells. In this small series of specimens, we did not observe this phenomenon. Further, we believe that the excision of this minimal inflammatory rim will provide accurate excision of tumor tissue combined with inflammatory tissue.
This study shows that generic tumor characteristics, like extracellular acidosis, are able to distinguish between malignant and benign disease. Thus, it gives surgeons the opportunity to change their surgical plan during and directly after surgical tumor excision. This direct feedback mechanism has the potential to decrease the amount of tumor positive margins. Nevertheless, the direct effect of this intervention in terms of morbidity, mortality and survival, needs to be studied in great randomized controlled trials in a major number of patients. A subsequent phase II study is currently performed in the United States of America, investigating the possibilities of visualization of novel solid tumors like prostate cancer and ovarian cancer. However, a shorter tracer administration and imaging interval is being investigated in the ongoing Phase II study, since administration only a few hours prior to surgery might be preferable in terms of clinical implementation.
Case summary: pH probes in tumor targeted therapy
Together with our client TRACER evaluated the safety, pharmacokinetics and feasibility of the pH-probe in image-guided surgery, occult tumor detection and visualizing tumor borders in four different cancer types. The results showed that the use of physiologic parameters such as pH, which were previously inaccessible therapy targets, can become clinically relevant when used for tumor targeted therapy. We believe that the general implementation of fluorescence-guided surgery into standard clinical care can improve surgical decision making in a variety of surgical sub specialisms. However, further research to investigate the optimal generic tracer and standardized imaging protocols are needed to make surgical optical imaging a genuine success.
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- Harlaar NJ, Koller M, de Jongh SJ, van Leeuwen BL, Hemmer PH, Kruijff S, et al. Molecular fluorescence-guided surgery of peritoneal carcinomatosis of colorectal origin: a single-centre feasibility study 2016;1:283–90. https://doi.org/S2468-1253(16)30082-6 [pii].
- Koller M, Qiu SQ, Linssen MD, Jansen L, Kelder W, de Vries J, et al. Implementation and benchmarking of a novel analytical framework to clinically evaluate tumor-specific fluorescent tracers 2018;9:3739-018-05727-y. https://doi.org/10.1038/s41467-018-05727-y [doi].
- Voskuil FJ, de Jongh SJ, Hooghiemstra WTR, Linssen MD, Steinkamp PJ, de Visscher SAHJ, et al. Fluorescence-guided imaging for resection margin evaluation in head and neck cancer patients using cetuximab-800CW: A quantitative dose-escalation study 2020;10:3994–4005. https://doi.org/10.7150/thno.43227 [doi].
- Voskuil FJ, Steinkamp PJ, Zhao T, van der Vegt B, Koller M, Doff JJ, et al. Exploiting metabolic acidosis in solid cancers using a tumor-agnostic pH-activatable nanoprobe for fluorescence-guided surgery 2020;11:3257-020-16814–4. https://doi.org/10.1038/s41467-020-16814-4 [doi].