Ovarian cancer is the leading cause of death among women with gynecologic malignancies.1 In dogs, the prevalence of ovarian cancer is low and ranges from 0.5% to 6.2%; however, the true rate of ovarian cancer in dogs is likely underestimated because many female dogs undergo elective ovariohysterectomy or oophorectomy at a young age for the purpose of sterilization.2 Ovarian tumors in dogs are histologically similar to those in women; therefore, dogs may serve as a useful species for investigating ovarian neoplasia in women.3 Typically, in both dogs and women, ovarian cancer is highly malignant and metastatic and often remains undetected until the patient is at an advanced stage of the disease.
In women, complete cytoreductive surgery to eliminate all gross residual disease is the treatment of choice for advanced ovarian cancer and has been shown to dramatically improve the likelihood of survival.4–6 Cytoreductive surgery consists of extirpation of the primary ovarian tumor as well as any metastatic tumors. Although minimally invasive cytoreductive surgical techniques have been described,7 cytoreduction of ovarian tumors is typically accomplished by sharp dissection and conventional electrocautery, which may lead to hemorrhage and associated complications in women. In veterinary medicine, laparoscopic techniques for oophorectomy and ovariohysterectomy in dogs have also been well described, and those minimally invasive methods are associated with lower morbidity rates and faster recovery times, compared with traditional open surgical methods.8–10 However, complications can develop following minimally invasive oophorectomy and ovariohysterectomy procedures,11,12 and efforts to develop less invasive alternatives for surgical sterilization of female dogs continue.13–15
In women with stage II/III hormone receptor-positive breast cancer, ovarian ablation with goserelin significantly improves survival times.16 Hyperthermic ablation technology, such as MWA, has evolved rapidly over the past 30 years and is commonly used in human oncology for minimally invasive percutaneous treatment of primary and metastatic tumors.17 Ablation is considered a first-line treatment and is preferred over surgical resection for small hepatocellular carcinomas in human patients with preserved liver function who are not transplant candidates.18 In dogs, MWA has been used for the treatment of hepatic neoplasia19,20 and ablation of pulmonary metastases via thoracoscopy.21 Benefits of percutaneous hyperthermic ablation therapy relative to surgical resection include decreases in morbidity, hemorrhage, duration of hospitalization, and cost.22–26 Compared with radiofrequency ablation, MWA has several advantages including a smaller probe diameter, faster ablation times, more versatile energy deposition, reduced heat-sink effects, and the ability to perform multiple synchronous ablations.27–31
Results of PubMed searches conducted on August 1, 2017, and July 1, 2018, with the search parameters “microwave ablation” and “ovarian” or “ovaries” suggested that MWA of ovarian tissue has not been described in dogs or human patients. In addition to the treatment of ovarian neoplasia, potential applications of MWA in regard to ovarian tissue include elective sterilization and treatment of hyperplastic (cystic) conditions in both veterinary and human patients. The objectives of the study reported here were to determine the optimal MWA energy profile (ie, duration of ablation required at an emission power of 45 W) necessary for complete destruction of clinically normal canine ovarian tissue as determined by histologic assessment of cell viability and to evaluate the efficacy and feasibility of the use of percutaneous ultrasonography and laparoscopy to guide MWA of ovarian tissue in dogs.
Materials and Methods
Animals
The study consisted of ex vivo and in vivo trials, and all study procedures were reviewed and approved by the University of Florida Institutional Animal Care and Use Committee. Twenty-two client-owned female dogs that weighed > 10 kg and were presented to the veterinary teaching hospital for routine gonadectomy were enrolled in the study. All dogs were considered healthy on the basis of results of a physical examination and evaluation of PCV and total solids, blood glucose, and BUN concentrations. Owner consent was obtained for each dog prior to study enrollment.
Ex vivo trial
Thirteen dogs were enrolled in the ex vivo trial. The method used for oophorectomy was chosen at the discretion of the surgeon and owner. All dogs were anesthetized and prepared for the selected procedure in a routine manner. For 11 dogs, oophorectomy was performed through a ventral midline celiotomy (open method). Each ovary was located and retracted caudally, after which the suspensory ligament was stretched or broken to improve visual observation of the procedure. The ovarian pedicle, which consisted of the ovarian artery and vein, suspensory ligament, and mesovarium, was ligated with absorbable monofilament suture and transected. The uterine tube, mesosalpinx, and uterine artery and vein were subsequently ligated and transected at the cranial aspect of the uterine horn, and the ovary was removed from the abdominal cavity. For the remaining 2 dogs, oophorectomy was performed laparoscopically by use of a 2-port technique with a vessel-sealing device used for vessel and tissue ligation and transection. The ligation and transection sites were the same for both the open and laparoscopic methods. All incisions were closed in a routine manner, and the dogs were allowed to recover from anesthesia.
All measurements and ex vivo ablations were performed by 1 investigator (VDV). For each ovary, the time at which the first clamp or vessel-sealing device was applied to the ovarian pedicle intraoperatively was recorded. After the ovary was removed from the patient, it was isolated from the ovarian bursa, and the maximum height, width, and length were measured with a digital caliper. An ellipsoid equation was used to calculate the volume of the ovary as follows: 4/3 × 3.14 × length × height × width. For the first 3 dogs, 1 ovary remained unablated and was processed in parallel with the ablated ovary to serve as a histologic control so that the ablation-induced ischemic effects could be assessed.
A 17-gauge MWA probea was used to perform MWA on each designated ovary. Prior to each use, the probe was tested for dysfunction in accordance with the manufacturer's recommendations. This test was performed by submerging the probe in saline (0.9% NaCl) solution, initiating test ablation, and observing for bubbles. The formation of bubbles would indicate leakage of gas through a physical defect in the probe.
Ovaries were randomly assigned by means of a random number generator to undergo MWA at an emission power of 45 W for 60 (group 1; n = 11) or 90 (group 2; 12) seconds. The MWA settings were chosen on the basis of institutional experience and optimal settings observed for hepatic ablation.19,20 For each ovary, the MWA probe was inserted in the same orientation and the emission zone was completely concealed within the ovarian tissue. The probe temperature was determined continuously by the MWA system. Ablation time and maximum temperature were documented for each procedure. After each procedure, the MWA probe was inspected grossly for damage and cleaned with an enzymatic solution.b The ovaries were submitted to the pathology laboratory where they were sectioned, snap frozen in liquid nitrogen, and stored at −80°C until processed for histologic examination. The time when tissue specimens were frozen was recorded.
In vivo trial
MWA and laparoscopic oophorectomy—Nine dogs were enrolled in the in vivo trial. All percutaneous laparoscopic MWA and oophorectomy procedures were performed by the same board-certified veterinary surgeon (JBC). Each dog was anesthetized, positioned in dorsal recumbency, and aseptically prepared for surgery in a routine manner.
For the first dog, a 4- to 9-MHz microconvex array ultrasound probe was used to image the ovaries and adjacent structures in an attempt to establish a safe window to percutaneously insert the 17-gauge MWA probe under ultrasound guidance. After insertion of the MWA probe, the left ovary was ablated at 45 W for 90 seconds under ultrasound visualization. Ablation time and the maximum temperature achieved during ablation were documented. Following ablation of the left ovary, a safe corridor for ultrasound-guided ablation could not be established for the right ovary. The subject was repositioned from dorsal recumbency to an oblique left lateral position, but a safe corridor for ablation could still not be established. Microwave ablation of the right ovary was achieved under laparoscopic guidance. Following completion of MWA, the dog underwent a routine laparoscopic oophorectomy. The initial ovary that underwent ultrasound-guided MWA was excluded from all analyses.
Computed tomography guidance and ultrasound-guided ovarian hydrodissection were attempted in the subsequent dog but also failed to provide a safe corridor for percutaneous MWA. The ovaries of that dog underwent MWA under laparoscopic guidance, and laparoscopic oophorectomy was performed following completion of MWA. Percutaneous ultrasound-guided MWA was not attempted in any of the other dogs.
For all laparoscopic procedures, a single multiaccess laparoscopic port was placed through the abdominal wall on the ventral midline at the level of the umbilicus. The abdomen was insufflated with carbon dioxide, and the patient was rotated into dorsolateral recumbency to expose the ovarian pedicle of interest. For laparoscopic-guided MWA, a 0° laparoscope was inserted through the port to visualize the ovarian pedicle. The proper ligament was grasped with laparoscopic forceps and elevated to stabilize the ovary. With laparoscopic guidance, the MWA probe was placed percutaneously through the ventrolateral abdominal wall at an orientation parallel to the long axis of the ovary. The ovarian bursa was not incised prior to MWA for the 3 initial ovaries treated but was incised (n = 3) or completely dissected (11) prior to MWA for the remainder of the ovaries to improve visualization of the ovary and accuracy of probe placement. After the probe was positioned within the ovary, the ovary was held approximately 3 cm from the abdominal wall and viscera and ablated at 45 W for 90 seconds, on the basis of results obtained during the ex vivo trial of the study. Following ablation of the ovary, a 5-mm bipolar energy devicec was used for vascular sealing and transecting the ovarian pedicle and proper ligament, and the time of ligation was recorded. The ovary was then removed with the laparoscopic port and immediately submitted to the pathology laboratory for processing and histologic examination.
After both ovaries were removed, the port incisions were closed in a routine manner, and the dogs were allowed to recover from anesthesia. All dogs were monitored for 24 hours after surgery and received hydromorphone (0.05 mg/kg, IV, q 4 to 6 h) as needed for analgesia. A validated pain-scoring method32 for dogs was used to assign a pain score to each dog every 4 hours. Any dog assigned a score > 1 was administered an additional dose of hydromorphone (0.05 mg/kg, IV) for rescue analgesia. All dogs were discharged from the hospital after return of bowel function and assignment of a pain score < 1, which was generally the day after surgery. Dogs were prescribed tramadol (3 to 5 mg/kg, PO, q 8 to 12 h for 5 days) to provide analgesia as needed at home. A classification scheme33 for surgical complications was used to characterize the safety of MWA in the in vivo trial.
Intraoperative laparoscopic videos were obtained for each dog and retrospectively reviewed by 2 investigators (JBC and VDV) who were unaware of the histologic outcome for all ovaries. The degree of exposure achieved for each ovary was subjectively assessed and categorized as complete or incomplete, as was the accuracy of MWA probe placement, which was categorized as axial (accurate) or abaxial (inaccurate).
Histologic evaluation—Immediately after each ablated ovary was removed from the abdominal cavity, the maximum length, width, and height of the organ were measured with a digital caliper, and the ovarian volume was calculated as described for the ex vivo trial. The macroscopic appearance of each ovary was described. Then, each ovary was cut into 3 standardized representative sections (1 transverse cross section and 2 transverse lateral sections). The sections were snap frozen in liquid nitrogen, and the time when sections were frozen was recorded. Sections were stored at −80°C until processed for histologic examination.
All frozen ovarian tissue specimens from the ex vivo and in vivo trials were processed for histologic examination in a routine manner and stained with H&E and NADH stains by 1 histologic technician. All sections were histologically examined by a board-certified veterinary pathologist (MJD). Each section was assessed for the presence of viable ovarian cells and extent of ablation, which was categorized as complete or incomplete.
Data analysis
Descriptive data were generated for the study subjects. The median and IQR were used to summarize continuous variables. For the ex vivo trial, the Wilcoxon rank sum test was used to compare ovarian volume and peak ablation temperature between groups 1 (ablation duration, 60 seconds) and 2 (ablation duration, 90 seconds). The Fisher exact test was used to compare the proportion of ovaries for which complete ablation was achieved between groups 1 and 2. For the in vivo trial, the Wilcoxon rank sum test was used to compare ovarian volume and peak ablation temperature between completely and incompletely ablated ovaries. Values of P < 0.05 were considered significant for all analyses.
Results
Ex vivo trial
The 13 dogs enrolled in the ex vivo trial included 7 mixed-breed dogs, 2 Great Danes, 1 German Shepherd Dog, 1 Golden Retriever, 1 Flat-coated Retriever, and 1 Labrador Retriever. The dogs had a median age of 17 months (IQR, 12 to 36 months) and weight of 23.5 kg (IQR, 20.1 to 30.8 kg).
A total of 26 ovaries were assessed during the ex vivo trial. Three ovaries did not undergo MWA and served as histologic controls. Eleven ovaries underwent MWA at 45 W for 60 seconds (group 1), and 12 ovaries underwent MWA at 45 W for 90 seconds (group 2). The median ovarian volume prior to MWA was 7.03 cm3 (IQR, 6.06 to 7.52 cm3) for the 3 control ovaries, 6.13 cm3 (IQR, 4.67 to 7.03 cm3) for group 1, and 6.12 cm3 (IQR, 4.16 to 8.61 cm3) for group 2. The median ovarian volume prior to MWA did not differ significantly (P = 0.93) between groups 1 and 2.
The peak temperature achieved during MWA was not recorded for 1 ovary in group 1 owing to an inadvertent error; however, that ovary was not excluded from the analysis. The median peak temperature achieved during MWA did not differ significantly (P = 0.41) between groups 1 and 2 and was 105°C (IQR, 98°C to 113°C) for all 22 ovaries for which it was recorded.
The median duration from ligation of the ovarian pedicle to MWA of the ovary was 17 minutes (IQR, 13 to 21 minutes). The median duration from ligation of the ovarian pedicle to freezing of the ovarian tissue sections was 23 minutes (IQR, 18 to 27 minutes). For 1 ovary in group 1, the duration between ligation of the ovarian pedicle and freezing of the ovarian tissue was > 30 minutes. Histologic examination of NADH-stained tissue sections from that ovary revealed viable cells, which were indicative of incomplete ablation. Therefore, that ovary was not excluded from the data analyses.
Macroscopic evaluation revealed that the ovarian tissue was firmer and had variable gray discoloration after MWA, compared with prior to MWA. Ovaries categorized as completely ablated appeared to have more extensive gray discoloration, compared with ovaries categorized as incompletely ablated; however, the extent of that discoloration was not consistent across all specimens. Histologic evaluation of NADH-stained tissue sections revealed a clear distinction between viable and nonviable tissue for all but 2 ovaries (Figure 1). Those 2 ovaries, both from group 2, had equivocal staining on NADH-stained sections, and it was not possible to determine whether viable cells were present. Tissue specimen handling and staining error were considered unlikely because the same experienced histology technician processed all specimens; therefore, those 2 ovaries were excluded from further analyses. No evidence of ablation was detected in the 3 control ovaries. The proportion of completely ablated ovaries in group 2 (10/10) was significantly (P = 0.035) greater than that in group 1 (6/11). Consequently, the group 2 MWA protocol (45 W for 90 seconds) was used for the in vivo trial.
In vivo trial
The 9 dogs enrolled in the in vivo trial included 4 mixed-breed dogs, 2 Australian Cattle Dogs, 1 Beagle, 1 Border Collie, and 1 Golden Retriever. The dogs had a median age of 12 months (IQR, 12 to 18 months) and weight of 15.8 kg (IQR, 13.5 to 18.0 kg).
All ovaries were ablated at 45 W for 90 seconds. The median peak temperature achieved during ablation was 112°C (IQR, 93°C to 118°C). The peak temperature achieved during MWA was not recorded for 1 ovary owing to an inadvertent error; however, that ovary was not excluded from the analysis. The median peak temperature did not differ significantly between completely ablated and incompletely ablated ovaries.
The median duration from sealing (ligation) of the ovarian pedicle to freezing of the ovarian tissue sections was 13 minutes (IQR, 11 to 16 minutes). The duration between sealing of the ovarian pedicle and freezing of the ovarian tissue was > 30 minutes for 1 ovary. Histologic examination of NADH-stained tissue sections from that ovary revealed viable cells, which were indicative of incomplete ablation. Therefore, that ovary was not excluded from the data analyses.
The median ovarian volume was 5.01 cm3 (IQR, 3.16 to 8.02 cm3). The median ovarian volume did not differ significantly between completely ablated and incompletely ablated ovaries.
Microwave ablation was performed percutaneously with ultrasonographic guidance for 1 ovary and resulted in incomplete destruction of the ovarian tissue. For the remaining 17 ovaries, MWA was performed with laparoscopic guidance and complete ablation of the ovarian tissue was achieved for 12 of those ovaries (Figure 2). Complete ablation was achieved for the final 8 ovaries in which the ovarian bursa was dissected completely and accurate positioning of the probe in the longitudinal axis of the ovary was confirmed. Review of the intraoperative laparoscopic videos obtained for each subject revealed that, for the 17 ovaries that underwent MWA with laparoscopic guidance, exposure (visualization) of the ovary during MWA was complete for 11 and incomplete for 6 (Figure 3). For 2 ovaries with complete exposure during MWA, abaxial (inaccurate) positioning of the MWA probe was confirmed. Those ovaries were the only 2 for which incomplete ablation was achieved among the final 11 ovaries that were completely dissected from the ovarian bursa prior to MWA.
Complications associated with MWA were all classified as grade 1 and included minor thermal injury of the body wall in the dog that underwent ultrasound-guided MWA of 1 ovary and minor bruising of the skin at the level of one of the MWA probe insertion sites in 2 dogs.
Discussion
The objectives of the study reported here were to determine the optimal energy profile (duration of ablation at an emission power of 45 W) required to completely ablate clinically normal ovaries in dogs and to assess the feasibility and efficacy of ultrasonographic and laparoscopic guidance for minimally invasive MWA of ovarian tissue to gain information and inform future investigations of the clinical applications of MWA for routine sterilization of dogs and treatment of ovarian tumors in both dogs and women. Results indicated that the optimal energy profile for MWA to achieve complete ablation of clinically normal canine ovaries was 45 W for 90 seconds. Additionally, results suggested that MWA with laparoscopic guidance was feasible and safe. However, ultrasound-guided MWA did not appear to be feasible and was abandoned in this study after use of that method resulted in incomplete ablation of 1 ovary in 1 dog.
Microwave ablation may provide specific advantages for the treatment of advanced ovarian cancer, compared with traditional open surgical approaches for extirpation of ovarian tumors by sharp dissection and electrocautery. Microwave ablation may be more tenable for treatment of tumors located within the bony pelvis or near the pelvic vasculature and abdominal wall. In women, the ovaries are easily accessible and readily visible by laparoscopy owing to the absence of an ovarian bursa, which obviates dissection of the mesovarium. Additionally, MWA causes thermal cell death and coagulative necrosis, which may limit hemorrhage and thereby be beneficial for cytoreduction of ovarian tumor metastases. Other modalities used for cytoreduction of ovarian cancer include argon laser (with a high-energy source [514 nm]), argon beam coagulator (with a high monopolar current of electrons), and neutral argon plasma energy; however, these modalities do not have the penetrating potential of microwave energy and are therefore used for the ablation of superficial and small-volume metastases, rather than solid organs.34,35
In the ex vivo trial of the present study, MWA at 45 W for 60 seconds resulted in variable incomplete ablation of ovarian tissue. Partial ablation of ovarian tissue may be desirable for certain hyperplastic conditions in women, such as polycystic ovary syndrome, for whom the preservation or recovery of fertility is prioritized. Incomplete ablation of ovaries was considered an undesirable outcome in the present study. Incomplete ablation was attributed to an insufficient duration of MWA, which was confirmed by the consistent complete ablation of ovaries that underwent MWA at 45 W for 90 seconds.
Use of a percutaneous, ultrasound-guided approach for MWA was performed for 1 ovary in the in vivo trial of the present study but was deemed unsafe because of the risk for inadvertent damage to vital anatomic structures, such as the kidneys and bowel, located in close proximity to the ovaries. For the dog in which MWA with ultrasonographic guidance was attempted, a safe corridor for MWA was thought to be present for the left but not the right ovary. However, during the laparoscopic oophorectomy procedure performed immediately after MWA of the left ovary, a minor thermal burn was identified on the body wall adjacent to the left ovary. Fortunately, further laparoscopic exploration of the area revealed no other injuries. Limitations of ultrasound-guided MWA for ovarian ablation were recognized a priori; however, we believed MWA of ovaries with ultrasonographic guidance was worthwhile in the present study to reaffirm technique allocation for future studies. Conversely, in this study, use of laparoscopic guidance for MWA of ovaries was feasible, safe, and effective and may be a useful adjunct for laparoscopic gynecologic surgery.
In the present study, macroscopic changes were variable and inconsistent between completely and incompletely ablated ovaries; therefore, macroscopic evaluation should not be used to assess ovarian tissue for completeness of ablation. The ovarian tissue specimens of the present study were stained with H&E and NADH stains for histologic examination because histologic examination of H&E-stained tissue sections is insufficient for evaluation of cell viability after ablation.36,37 However, histologic examination of H&E-stained tissue sections is valuable for evaluation of pathological lesions in areas with NADH stain uptake indicative of cell viability. The underlying cause for the equivocal interpretation of cell viability for 2 NADH-stained ovarian tissue specimens could not be determined but might have been caused by an error in sample processing. Regardless, the presence of viable tissue in those 2 specimens could not be ruled out.
The present study had multiple limitations, with the primary ones being the small sample size and evaluation of only 2 different settings for MWA. It is likely that use of alternative power settings and durations for MWA would have been effective and might indeed be required for ablation of large ovarian tumors. We did not evaluate human ovaries in the present study; however, the median volume for the clinically normal canine ovaries of this study was similar to the volume of clinically normal ovaries in women.38 Another limitation of the present study was that the ovarian tissue specimens were snap frozen within minutes after MWA; therefore, only direct effects of thermal injury were evaluated. Direct thermal injury of tissue encompasses changes observed immediately after an increase in temperature and results from a combination of biological and vascular mechanisms. Indirect thermal injury is manifested by progressive tissue damage after the hyperthermic stimulus is removed. Thus, it is possible that the viable cells observed in some incompletely ablated ovaries may have been irreversibly damaged and died over time. However, leaving incompletely ablated ovaries in situ in dogs could result in signs of estrus and potential pregnancy, and for patients with ovarian cancer, the presence of viable malignant cells could lead to local recurrence or metastasis.
Only clinically normal ovaries were assessed in the present study. Further investigation is necessary to evaluate the efficacy of MWA for cytoreduction of neoplastic or cystic ovaries. It is possible that MWA at a higher emission power and for a longer duration than the optimal MWA energy profile (45 W for 90 seconds) identified in this study will be required for ablation of neoplastic or cystic ovaries owing to alterations in both tissue composition and volume. Also, MWA with multiple simultaneously powered antennas may be an effective alternative for large tumors and warrants further investigation.
Results of the present study suggested that MWA at 45 W for 90 seconds was sufficient for complete ablation of clinically normal canine ovaries with volumes that ranged from 4.02 to 8.14 cm3. The use of MWA at 45 W for 60 seconds resulted in variable and incomplete ablation of clinically normal canine ovaries. Additionally, the use of ultrasonographic guidance for MWA of canine ovaries was not feasible, whereas the use of laparoscopic guidance of MWA was feasible, safe, and facilitated accurate positioning of the MWA probe within the ovary. Aside from being a feasible method for minimally invasive sterilization of dogs, laparoscopic-guided MWA may be useful for the treatment of hyperplastic or neoplastic ovarian conditions in dogs and women.
Acknowledgments
All described procedures were performed at the University of Florida College of Veterinary Medicine.
This manuscript represents a portion of a thesis submitted by Dr. Verpaalen to the Department of Small Animal Clinical Sciences, College of Veterinary Medicine, University of Florida as partial fulfillment of the requirements for a Master of Science degree.
Supported by a Consolidated Faculty Research Development Award Grant from the University of Florida College of Veterinary Medicine. NeuWave Medical, Madison, Wis, provided the microwave probes used in the study. The funding sources had no involvement in the study design, data analysis and interpretation, or writing and publication of the results for this project.
The authors declare that there were no conflicts of interest.
Presented as a poster presentation at the American College of Veterinary Surgery Summit, Phoenix, October 2018.
The authors thank Cat Monger and Melissa Brown for technical assistance.
ABBREVIATIONS
IQR | Interquartile (25th to 75th percentile) range |
MWA | Microwave ablation |
NADH | Nicotinamide adenine dinucleotide diaphorase |
Footnotes
Certus PR15 Ablation Probe, NeuWave Medical, Madison, Wis.
Bio-zyme, Osceola Supply, Tallahassee, Fla.
Ligasure, Covidien, Saint-Laurent, QC, Canada.
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