Ultrasonography of Osteosarcoma in Dogs
Sonography Atlas of Dogs

Abstract

Figure 2: In a panel of cell lines inclusive of known positive secretory controls (far left) and 5 osteosarcoma cell lines (human and dog), active secretions of (A) NGF, (B) ET‐1, and (C) PGE2 into the cell culture supernatants are quantified by ELISA after 48 hours of incubation and normalized for differences in metabolic activity.

Nociceptive Ligand Expression in Canine OS

All tumor samples demonstrated diffuse cytoplasmic staining for all 3 nociceptive ligands (Fig 3A). Among the 3 nociceptive ligands, ET‐1 was most robustly and consistently expressed with 16/16 samples demonstrating a 3+ staining score, whereas NGF and mPGES‐1 staining was 3+ positive in 9 and 10 samples, respectively (Fig 3B). Although all nociceptive ligands demonstrated cytoplasmic subcellular localization, immunohistochemical detection of mPGES‐1 also was identified within the nucleus in approximately 10–15% of malignant osteoblast cells.

Figure 3: (A) Microscopic appearance of a representative spontaneous canine OS sample by H&E staining and confirmed protein expressions of NGF, ET‐1, and mPGES‐1 by immunohistochemistry. (B) Tabulated immunohistochemical staining summary for NGF, ET‐1, and mPGES‐1 expression in 16 spontaneously arising OS samples.

Cancer Pain and Circulating Nociceptive Ligands in Canine OS

In 10 untreated dogs with OS (Table S1), serial changes in VAS pain scores and circulating nociceptive ligand concentrations were characterized after effective pain alleviation. The median pretreatment (day 0) VAS pain score was 3.5 (range, 0.0–7.0), which decreased after the institution of treatment to 1.5 (range, 0.0–6.0) at day 28 and 2.5 (range, 0.0–6.0) at day 84, and subsequently increased to 6.0 (range, 1.0–9.0) at time of bone pain progression (Fig 4A). Median VAS pain scores on days 28 (P < .05) and 84 (P < .05) were significantly decreased in comparison with day 0.

Figure 4: Serial changes as a function of time in (A) VAS pain scores and plasma concentrations of (B) NGF, (C) ET‐1, and (D) PGE2 derived from 10 OS‐bearing dogs treated with standardized palliative therapies. Data expressed as median and interquartile range for (A–C) and mean ± SEM for (D). Significant differences relative to Day 0 denoted with either *P < .05 or **P < .01.

In the same 10 dogs, circulating NGF, ET‐1, and PGE2 concentrations were serially quantified. In 7 dogs, NGF concentrations were below the limits of detection (Table S1), but the remaining 3 dogs had quantifiable NGF concentrations with median values of 132.6 pg/mL (range, 59.3–228.2), 152.6 pg/mL (range, 44.0–187.4), 139.6 pg/mL (range, 21.6–142.1), and 128.0 pg/mL (range, 24.6–174.4) at days 0, 28, 84, and failure, respectively (Fig 4B). Based on these 3 dogs, NGF concentrations did not appear to change after institution of effective palliative treatment, but statistical analysis was not performed because of the limited sample size. For ET‐1, 7 dogs had ET‐1 concentrations between 4.0 and 12.0 fmol/mL, whereas 3 dogs had higher concentrations of ET‐1 ranging from 45.0 to 84.5 fmol/mL (Table S1). The median concentrations of ET‐1 at days 0, 28, 84, and failure was 8.8 fmol/mL (range, 4.0–84.5), 6.6 fmol/ml (range, 4.4–62.6), 6.5 fmol/mL (range, 4.0–60.3), and 9.2 fmol/mL (range, 4.3–57.3), respectively. Compared to pretreatment (day 0), ET‐1 concentrations were significantly decreased on day 84 (P < .05; Fig 4C). The average plasma concentrations of PGE2 were 368.7 ± 73.5 pg/mL, 134.1 ± 20.5 pg/mL, 122.7 ± 17.5 pg/mL, and 129.7 ± 11.8 pg/mL on days 0, 28, 84, and failure, respectively (Fig 4D). Decreases in plasma PGE2 concentrations in comparison with day 0 were significant for all time points: day 28 (P < .01), day 84 (P < .01), and failure (P < .01).

No correlations between individual VAS pain score and ET‐1 or PGE2 concentrations at each respective time point (days 0, 28, 84, and failure) were identified. The ability to assess for any correlation between individual VAS pain score and NGF concentrations was precluded given the limited number of dogs (n = 3) with measurable NGF concentrations. Relative primary bone tumor burden in dogs with OS was expressed as ATV, and among the 10 OS‐bearing dogs was calculated to be 107.0 ± 13.6 cm³. No correlation between ATV and any individual pain parameter at baseline was identified.

Disorganized Foci of Nerve Ending Proliferations in Canine OS

To investigate if sensory nerve fiber sprouting occurs in canine OS, 10 archived primary OS samples were stained for 2 neuronal markers: class III β tubulin and peripherin (Fig 5A,B). Systematic evaluation of 5 microscopic fields (135,000 μm²/field) containing periosteal tissues adjacent to viable malignant osteoblasts identified positive staining for both class III β tubulin and peripherin in 20% of tumor samples (Fig 5C,D), whereas periosteum from normal bone (data not shown) and the majority of OS samples did not show any significant immunolabeling (Fig 5E,F). Quantitatively, in 2 of the 10 OS samples, the number of positively labelled nerve foci per microscopic field ranged from 0 to 25, with a median value of 3 nerve termini/field. In the remaining 10 bone samples (8 OS and 2 normal bone), no obvious detectable nerve termini were identified within the periosteum.

Figure 5: Microscopic appearance of a peripheral sensory nerve traversing through control tissue as indicated by positive immunohistochemical staining for (A) class III β tubulin and (B) peripherin. Haphazard and disorganized proliferation of neuronal endings identified within OS‐associated periosteum as determine by positive immunohistochemical staining for (C) class III β tubulin and (D) peripherin. Representative OS‐associated periosteum which is devoid of excessive neuronal end proliferations based upon the absence of (E) class III β tubulin and (F) peripherin immunohistochemical staining.

Discussion

Although various nociceptive ligands likely participate in generating OS bone cancer pain, the current investigation focused on NGF, ET‐1, and PGE2 given their potential roles in not only malignant osteolytic nociception but also OS protumorigenic biology. In particular, NGF and ET‐1 have been intensively evaluated as drug targets for the alleviation of bone cancer pain in murine preclinical models. In these studies, the neutralization of NGF with blocking antibodies or small molecule inhibitors decreased nociceptive guarding behaviors in mice implanted with prostatic carcinoma or osteolytic sarcoma cells within the bone medullary cavity.21, 22, 23 Similarly, ET‐1 signaling blockade with small molecule inhibitors in tumor‐bearing mice alleviated painful sensations induced by the osteolytic growth of prostatic carcinoma or bone sarcoma cells.24, 25 Although inflammatory eicosanoids, particularly PGE2, have long been recognized to participate in nociceptor sensitization associated with inflammatory disorders, recent investigations have directly linked PGE2 with tumor‐induced osteolysis and bone cancer pain.26, 27, 28 Importantly, the rate limiting enzyme involved in PGE2 generation (mPGES‐1) has been demonstrated to enhance bone tumor growth and malignant osteolytic pain behaviors in preclinical murine models,29 and provides a biologic rationale for inhibiting mPGES‐1 enzymatic activities to ameliorate pain associated with malignant osteolysis. Despite many studies defining the drivers of malignant bone pain in murine preclinical models, canine OS‐associated bone pain remains incompletely defined and warrants studies to identify pathways involved in the development and maintenance of bone cancer pain in dogs.

Although localized pain is a salient hallmark in dogs diagnosed with OS, the clinical spectrum of pain symptomatology is heterogeneous among affected patients, and likely reflects the complex interplay among several tumor‐ and host‐related factors including degree of osteolysis, tumor volume and location, skeletal mass and physical activity. Findings in this study suggest that additional tumor‐specific factors also could contribute to the variable spectrum of malignant bone pain, specifically the inherent capacity of OS cells to express and actively secrete nociceptive ligands. Malignant osteoblast cell lines demonstrated a wide variability in the secretion of nociceptive ligands, with the magnitude of difference between the highest and lowest secreted concentrations ranging approximately 10‐fold, 100‐fold, and 800‐fold for NGF, ET‐1, and PGE2, respectively. In addition, all naturally occurring OS samples demonstrated uniform and positive staining for NGF, ET‐1, and mPGES‐1, and if malignant osteoblasts retain divergent capacities for localized nociceptive ligand secretion within the bone tumor microenvironment, it is plausible that the severity of bone pain in dogs could be partially mediated by the existence of different clonal populations with variable secretory capacities.

In this study, the relationship between VAS pain scores and plasma concentrations of NGF, ET‐1, and PGE2 were explored in OS‐bearing dogs receiving standardized palliative treatment. As expected, the alleviation of pain after effective palliative treatment decreased the median VAS pain scores on days 28 and 84 in comparison with day 0 and, upon recurrence of breakthrough pain, VAS pain scores rebounded back to pretreatment levels. Plasma concentrations of NGF, ET‐1, and PGE2 tended to decrease after palliative treatment, but strong correlations between VAS pain scores and circulating nociceptive ligand concentrations were not identified in the limited population of dogs evaluated. As such, the preliminary data generated from the current investigation do not support the utility of plasma NGF, ET‐1, and PGE2 concentrations as surrogates of bone pain or absolute tumor volume in dogs with OS. Corroborating the imperfect association between pain intensity and circulating nociceptive ligands identified in this study, correlations between plasma NGF concentrations and pain intensity in people suffering from various pathologic conditions including chronic migraine, interstitial cystitis, and temporomandibular joint and myofascial pain also have yielded conflicting results.30, 31, 32

Although circulating concentrations of NGF, ET‐1, and PGE2 were not correlated with pain status in dogs with OS, ours is the first report to serially characterize pre‐ and posttreatment plasma concentrations of nociceptive ligands in dogs with OS. For NGF and ET‐1, it is noteworthy that systemic concentrations of both ligands could be dichotomously categorized as being either marginally or robustly detectable, and the observed pattern could be consistent with differing secretory capacities of canine OS cells as demonstrated in vitro. Among the 3 ligands, plasma PGE2 concentrations were most consistently detectable in dogs with OS, and demonstrated expected decreases after administration of nonsteroidal anti‐inflammatory drugs (NSAIDs), findings consistent with historical studies in tumor‐bearing dogs treated with NSAIDs.33

Gradual intensification of bone pain is a frequent clinical observation in dogs diagnosed with OS, and is a consequence of progressive mechanical, chemical, and neuronal perturbations within the immediate bone tumor microenvironment. In murine preclinical models of malignant bone pain, aberrant nerve sprouting has been identified as a driver of pathologic pain intensification,34, 35 yet whether an analogous process is operative in naturally occurring bone tumors such as canine OS has not been reported previously. Of 10 primary OS samples, excessive proliferation of nerve endings within tumor‐associated periosteum was definitively observed in 2 cases, suggesting that aberrant nerve sprouting might contribute to progressive pain intensification in some affected dogs. Given that only a small fraction of the periosteal surface area was microscopically evaluated in this study, the percentage of bone samples recognized to exhibit aberrant nerve sprouting likely underestimates the true frequency of neuropathologic alterations induced by OS‐associated chronic bone pain. Based upon these findings, it would be reasonable to suggest that treatment strategies that inhibit pathologic neuronal sprouting, such as NGF blockade, might attenuate the severity of bone pain in dogs with OS.

Despite the novel findings of this study, there are several investigative limitations that should be recognized. First, the mere demonstration of nociceptive ligand expression by OS cells does not provide evidence for the direct or indirect participation of OS cells in pain generation, rather it can only be stated that OS cells are capable of producing ligands with recognized nociceptive functions. Second, although NGF, ET‐1, and PGE2 were chosen for investigation based on their known involvement in nociception, these molecules can be produced by a broad range of cell types and also exert diverse effects completely unrelated to pain sensation. In fact, within the bone tumor microenvironment, nonmalignant resident cells including osteoclasts, mature osteocytes, and trafficking immune cells were identified to stain positively for some of the nociceptive ligands characterized. Hence, their evaluation in dogs with OS as exclusive biomarkers of bone pain would be erroneous given their involvement in diverse homeostatic processes including cardiovascular, neuronal, and cell membrane physiology. Third, rather than measuring systemic circulating nociceptive ligands, assessment of their intratumoral concentrations would have been expected to correlate better with localized bone pain status, as supported by preclinical murine tumor models.28

In summary, the descriptive findings reported here provide incremental advancements in identifying potential driver pathways that might participate in malignant bone pain generation in dogs with OS. Improving treatment outcomes in people suffering from bone cancer pain has been possible only by the initial identification of key cellular processes involved in malignant osteolysis and pathologic nociceptor activation, with several novel analgesic targets being validated and consequently translated into clinical practice.24, 36, 37 The rational development and clinical institution of superior analgesic regimens for dogs suffering from OS‐associated bone pain will require a similar discovery path. The findings of thist study might serve as an initial catalyst to stimulate future research into the functional pathways responsible for pathologic nociception in dogs suffering from malignant bone pain.

Acknowledgment

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