CORR® Tumor Board: Micrometastatic Drug Screening Platform Shows Heterogeneous Response to MAP Chemotherapy in Osteosarcoma Cell Lines

What are the surgical and research implications of this study?

Megan E. Anderson MD

Orthopaedic Oncology Surgeon

Beth Israel Deaconess Medical Center and Boston Children’s Hospital

Targeted therapy is a common catchphrase in orthopaedic oncology, and for good reason. Thanks to recent advancements in how we define gene mutations and specific driver genes in tumors, researchers now can develop and use agents that target and kill abnormal cells, leaving normal cells unharmed. Pembrolizumab (the anti-PD-1 monoclonal antibody) for patients with advanced nonsmall cell lung cancer with a high level of programmed death ligand 1 (PD-L1) [8] and vemurafenib and dabrafenib (BRAF kinase inhibitors) in patients with advanced melanoma with BRAF mutations [6] are just two examples of effective targeted therapies in oncology.

But not all targeted therapies are effective. Studies on targeted therapy in osteosarcoma have been disappointing [10]. Osteosarcomas have complex and multiple gene mutations and are heterogeneous not only among patients, but also in primary versus metastatic tumors in the same patient. When we do identify common mutations, it is generally unclear which one is the “driver” mutation that has the greatest effect on the malignancy of a tumor.

But current research focusing on the metastatic potential of osteosarcoma and targeting those mechanisms has shown some promise. The current study by Collier and colleagues [3], for example, represents important lab research that has the potential to translate to effective novel agents in osteosarcoma. Theoretically, the genomic profile of a tumor and a specific cell line could be developed rapidly from a biopsy specimen from a patient’s tumor [2]. Sarcospheres (osteosarcoma stem cells grown as clones in a spherical conformation) derived from that patient’s specific cell line could then be used to test standard chemotherapy as well as novel agents against it. Patients could then be offered enrollment in large multinational studies with eligibility determined by demonstration of response to an investigational therapy using the screening described in the current study. If the agents in a particular study are tailored only to patients with tumors that have a higher likelihood of responding, the pace and efficacy of drug development in osteosarcoma would be vastly improved. We are in the infancy of such investigation, but the efforts of teams like that in the current study show promise that targeted therapy may be possible in osteosarcoma in the future.

What issues does this study raise in terms of musculoskeletal imaging?

Jim S. Wu MD

Musculoskeletal Radiologist

Beth Israel Deaconess Medical Center

I read the study by Collier and colleagues [3] with great interest as it challenges radiologists like myself to develop new imaging techniques as they relate to personalized medicine. The authors of the current study generate sarcospheres from highly metastatic osteosarcoma cell lines as a way to test current and potentially new therapeutic agents. This study is a stepping stone towards developing cell lines from a patient’s individual tumor by which ideal therapeutics can be identified. Similar studies could challenge radiologists to develop imaging tests that can be personalized to individual patients.

Currently, the two main imaging goals for patients with osteosarcoma are: (1) To delineate the full extent of the primary tumor to aid in surgical resection and (2) to identify sites of distant spread before and after therapy.

For the first goal, radiographs and CT are the best imaging modalities to assess for osseous changes, and MRI is the preferred method for delineating any soft tissue and marrow abnormalities. With recent advances in CT and MRI sequences, this first goal is typically met.

Current imaging tests can generally achieve the second goal, but only for macroscopic disease. Overall survival for osteosarcoma has not considerably changed over the past few decades, partly due to a lack of new effective therapeutics, as Collier and colleagues [3] highlight in their study. However, the lack of progress in this matter is also due to our inability to identify microscopic tumor spread; which could potentially be solved with personalized imaging. As with many malignancies, the earlier we can detect disease and treat it, the better the prognosis.

Detecting metastatic disease in osteosarcoma is typically performed with a variety of whole body nuclear medicine studies, but is limited to disease that we can physically see, or is radiologically visible. Generally, lesions smaller than 1 cm are not detected. Bone scintigraphy uses the radiotracer technetium 99m-methyl diphosphonate (Tc-99m MDP), which attaches to hydroxyapatite in bone and accumulates at sites of osteoblastic activity and high bone turnover. It has high sensitivity, but low specificity as other conditions such as fracture can also have radiotracer uptake as well. Positron emission tomography/CT (PET/CT) using either ¹⁸F-Sodium fluoride (NaF) or ¹⁸F-fluorodeoxyglucose (FDG) radiotracers can detect metastatic disease in osteosarcoma. NaF goes to sites of osteoblastic activity, and FDG is radiolabeled glucose and goes to tissue that needs a carbon backbone and energy from glucose. Although both take advantage of different aspects of osteosarcoma physiology, neither test is specific for osteosarcoma.

Luckily, there are new tests on the horizon that hold promise, although not quite yet to the extent of personalized imaging. Recently, a bisphosphonate MR contrast agent was developed that can target the osteosarcoma primary tumor and lung metastases, as shown in mice [4]. Another exciting area is the use of targeted molecular imaging. CXCR4 is a receptor for the chemokine CXCL12 and is expressed in both primary and metastatic osteosarcoma. Using a CXCR4 targeted near-infrared fluorescent imaging agent, researchers have shown the feasibility to specifically target and image osteosarcoma metastases on a molecular level [5]. With continued advances in molecular imaging, ideally one can generate imaging agents specific to each patient’s individual tumor similar to what Collier and colleagues [3] hope to do with their sarcospheres. This could allow for earlier detection of osteosarcoma metastatic disease thereby improving outcome.

What more does the surgeon need to know about musculoskeletal pathology in order to get the most out of this study?

Sara O. Vargas MD

Staff Pathologist

Boston Children’s Hospital

Osteosarcoma is an excellent model for the current study because it is a tumor where local control for the primary tumor can usually be achieved surgically, and metastatic tumor seems the more-relevant target for assessing chemotherapy efficacy. Cells from metastases are biologically different from those in the primary tumor. The evolving understanding of the differences rests in large part on the emerging and increasing appreciation of heterogeneity within an individual tumor.

Heterogeneity within a tumor can occur at the histologic, genetic, or epigenetic level; it pertains not only to individual tumor cells but to their environment, which may include other tumor cells, vessels, inflammatory cells, stromal cells, growth factors, and all other factors that can influence cellular processes. In osteosarcoma, cell heterogeneity at the histological level is obvious (Fig. 1). We see great variation in the type of differentiation: Osteoblastic, chondroblastic, fibroblastic, and less frequently, other lines of differentiation can be seen. There can be great variation in extracellular matrix production—nuclei can be large or small, dense or “open”; they can show typical or atypical mitoses. Cytoplasm can be scant or abundant.

Fig. 1.

Histologically, this example of osteosarcoma shows variation in cell size (pleomorphism). There are abnormal mitotic figures, reflecting impaired cell division machinery. Cells vary with respect to their position near osteoclasts and osteoid matrix. Hematoxylin and eosin; original magnification, 600x. (Published with permission from Sara O. Vargas MD).

Genetic heterogeneity within a tumor refers to the coexistence of tumor cells having distinct genetic codes, in other words, different subclones within the same tumor. Malignant osteosarcoma cells are known for their increased tolerance for genomic instability. As the tumor grows, cells with dysfunctional DNA repair may have a survival advantage, resulting in a high rate of acquisition of mutations in successive generations of tumor cells [9]. Epigenetic heterogeneity refers to differently altered expression of the genetic code among tumor cells. Epigenetic states within a tumor vary, controlled by many different means including DNA methylation, histone modification, and chromatin remodeling. Some of these modifications affect a cell’s “stemness,” or ability to propagate [7]. There is increasing evidence to support the concept that a tumor, almost like a living organism, evolves subspecialized populations of cells that help support each other [1]. In glioblastoma, for example, it has been shown that different “driver” tyrosine kinase gene amplifications occur in cells located in close proximity to each other, in a seemingly organized arrangement [11]. Diverse cell populations may well coexist symbiotically in osteosarcoma, but this possibility is largely unexplored to date.

Genetic and epigenetic heterogeneity are highly interrelated with each other and with the cellular microenvironment or “soil.” With osteosarcoma, the lungs seem to provide a particularly hospitable environment for metastasis, for unknown reasons. Factors in the microenvironment that may be hospitable or inhospitable to tumor growth and progression might include, for example, oxygen tension, extracellular matrix composition, cell-derived vesicles (exosomes), soluble receptor tyrosine kinase ligands, and cytokine-induced survival signaling [7].

Tumor heterogeneity is of particular interest as a mechanism of resistance for targeted therapy. Emergence of therapy-resistant clones or persistence of subpopulations in a therapy-resistant microenvironment may be a consequence of tumor diversity.

With the above principles in mind, one can view the drug screening in the current study [3] as an important, but potentially incomplete, investigation into osteosarcoma chemosensitivity. The authors grew their sarcosphere cells from “highly metastatic tumor” that presumably included subpopulations from metastases, which are most likely to contain cells of relevance. However, it is impossible that a cell line would represent the breadth of cellular diversity inherent in an in vivo metastasis, and it certainly would not incorporate the microenvironmental factors encountered in vivo. Future studies more closely recapitulating in vivo conditions may be even more relevant.

Tumors in many ways may be as distinct as the individuals who carry them, and ultimately, tumor heterogeneity embodies both the challenge and the opportunity of “precision medicine.”