A 12.5-year-old 23 kg spayed female Labrador mix dog was referred to the neurology service at Purdue University, College of Veterinary Medicine, for potential hypophysectomy. Three months prior, the dog was diagnosed with hyperadrenocorticism by the referring veterinarian after a wellness visit revealed elevated liver enzymes and the owner reported an increased appetite. The patient's veterinarian performed an ACTH test (5ug/dl [1.0–5.0] for basal value and 25.1ug/dl [8.0–17.0] post-stimulation). An ultrasound performed by a board-certified radiologist revealed a diffuse increase in liver echogenicity and a bilateral adrenomegaly (0.75cm thickness for the right gland, 1.09 cm for the left gland, normal < 0.7cm). The dog was started on S-Adenosyl methionine (SAMe) 425 mg once daily and Lysodren (mitotane) 1mg/kg twice daily. The patient's cortisol values stabilized by three months after starting therapy (basal value 5.1ug/dl [1.0–5.0], post-stimulation value 10.8/dl [8.0–17.0]).
Neurology consultation.
Upon the first evaluation by Purdue's neurology service, the owner reported intermittent tremors of the pelvic limb (upper part, side non-specified) without any conscious abnormality. The owner also described stumbling or intermittent scuffling of the same leg. The patient had a persistent increased appetite and was regularly panting. Neurological examination was normal except for inconsistent positional ventral strabismus of the right eye. Upon physical examination, a small subcutaneous mass was present on the right flank, and a left caudal rear mass was previously diagnosed as papilloma. Biochemical analysis revealed elevated liver enzymes, hypercholesterolemia, hyperamylasemia, and hyperkalemia (Table 1). Hematology was unremarkable except for clumped platelets. Three survey thoracic radiographs were unremarkable except for the liver size extending beyond the coastal arch with rounded margins.
Table 1
Biochemical analysis. First biochemistry analysis. The letter “H” under the flag column represents a high value. The results, reference range, and unit measurements are provided for each test. (mg = miligrams, dl = deciliters, mmol = micromols, L = liters, g = grams, IU = international units, U = units, ALTv = alanine aminotransferase, GGT = gamma-glutamyl transferase).
Test | Result | Flag | Reference Range | Units |
Glucose | 102 | | 67–132 | mg/dL |
Blood Urea Nitrogen | 16 | | 7–32 | mg/d |
Creatinine | 0.80 | | 0.50–1.50 | mg/dL |
Phosphorus | 4.7 | | 2.2–7.9 | mg/dL |
Calcium | 11.0 | | 9.7–12.3 | mg/dL |
Sodium | 145 | | 138–148 | mmol/L |
Potassium | 5.3 | H | 3.5-5.0 | mmol/L |
Chloride | 114 | | 105–117 | mmol/L |
Carbon Dioxide | 23 | | 13–24 | mmol/L |
Anion Gap | 13.3 | | 9.0–18.0 | mmol/L |
Total Protein | 6.9 | | 4.8–6.9 | g/dL |
Albumin | 4.0 | H | 2.3–3.9 | g/dL |
Globulin | 2.9 | | 0.7–3.8 | g/dL |
ALTv | 187 | H | 3–69 | U/L |
Alkaline Phosphatase | 522 | H | 20–157 | IU/L |
GGT | 46 | H | 5–16 | IU/L |
Total Bilirubin | 0.50 | | 0.10–0.80 | mg/dL |
Cholesterol | 309 | H | 125–301 | mg/dL |
Amylase | 685 | | 378–1033 | IU/L |
Lipase | 1925 | H | 104–1753 | IU/L |
An MRI (GE 1.5T 8 channel-23x, Milwaukee, WI) exam was performed under general anesthesia. A board-certified veterinary radiologist identified a T2W hyperintense, T1W hyperintense mass (size length 16.2 mm x height 14.2mm, width 8mm) in the right cerebral hemisphere, within the right olfactory bulb area. The mass was characterized by strong homogeneous contrast enhancement, a broad-based contact with the falx cerebri, and a dural tail. Mild lysis of or pressure on the cribriform plate was suspected. The radiologist concluded the diagnosis of a possible meningioma of the right olfactory bulb (Fig. 1). In addition, the neurohypophysis was prominent and displaced to the right and caudally, creating suspicion for a microadenoma (size length 6 mm x height 5mm, width 5mm) (Fig. 1).
Conclusion of the first referral consultation.
The patient presented two synchronous brain tumors: a suspected right olfactory bulb meningioma without any obvious associated neurologic signs and a hypophyseal microadenoma associated with clinical signs of hyperadrenocorticism. Following a discussion with the owner and the concertation between internal medicine, neurology, and radiation oncology, radiation therapy was the elected treatment for both tumors. Before the radiation enrollment and with the imminent prospect of multiple anesthetic episodes, the dog underwent a cardiac examination with cardiac ultrasound; no cardiac abnormality was reported.
Radiation therapy.
Images acquisitions
To realize the treatment plan, the patient was positioned in a vacuum bag with her head immobilized by a bite block and thermoplastic mask (Civco Medical Solutions, Uniframe base plate, Orange City, IA) (Fig. 2). The mask position was indexed to the radiation table couch. Thin slices of 0.625 mm were acquired from our CT scan machine ( VCT 64-slices, GE Healthcare, Milwaukee, WI) without and with IV contrast injection (Ultrafast Bayer Healthcare, Wayne, NJ). The acquired images were transferred to our treatment plan, Eclipse (Varian Eclipse v11.0, Varian Medical Systems, Palo Alto). The precontrast series images were registered and fused with the MRI (GE 1.5T 8 channel-23x, Milwaukee, WI) T1 + contrast series images.
Radiation Plan Prescription
A definitive treatment with ablative intent (15 fractions of 3 Gy) was prescribed for both suspected tumors. The intracranial meningioma was treated with an intensity-modulated radiation therapy technique (IMRT), and a three-dimensional, four-field treatment was elected for the pituitary microadenoma. Both plans were delivered during the same anesthetic events.
Delivery
The patient was treated every other day with a 6MV linear accelerator (Varian 6EX, MLC Millenium 120 Dose Rate 400MU/mn (Varian Medical Systems, Palo Alto, CA)). The machine is equipped with record verification software (Aria, Varian Medical systems Palo Alto), and patient position accuracy was checked before delivery with port films (Kodak ACR-2000i -Oncocepts Rochester, NY). This patient setup has been previously reported by our institution [4].
For growth disease volume, the olfactory meningioma (m) and the pituitary microadenoma (p) were considered as separated growth target volumes (GTVm and GTVp) and contoured on the T1W + contrast series. For the meningioma, 3 mm margins were added to the GTVm: 2mm for microscopic disease and 1 mm for positioning uncertainty to get the planned target volume (PTVm). For the microadenoma, 2mm margins were added to obtain the pituitary planned target volume (PTVp). Critical structures were contoured separately (i.e., eyes, lens, optical nerves, optical chiasma, brain, cerebellum, brainstem, spinal cord, and ear bulla).
Two separate radiation plans were designed: 1) an IMRT for the meningioma and 2) a 3D conformal for the pituitary (Fig. 3). Each PTV dose was selected with the intent to receive more than 95% of the dose and less than 107%. A plan summation was realized to verify the cumulative dose and the separation between the two plans [5–7]. Before delivery, the IMRT treatment plan was reviewed by a physicist for quality insurance after comparing the planned fluence and the actual delivery fluence in a diode cylinder Arckcheck ™( Sun Nuclear, Melbourne, Florida). The plan was accepted as the difference in dose registered between the theoretical plan and the real plan delivered on the diodes cylinder matched. In other words, less than a 3% difference was observed for over 95% of the diode points selected (3mm distance). The patient received a total of 15 radiation sessions for both sites and recovered uneventfully from these treatments. She was discharged under prednisone 0.5mg/kg once a day to prevent brain radiation secondary effects, as well as S-Adenosylmethionine 425mg per day. The Lysodren 1mg/kg BID was continued.
Patient Follow-up.
The patient was rechecked at two weeks and one month after completion of the radiation treatment. The owner did not report any concern for appetite or water intake. Physical exams were unremarkable, and no radiation side effects were observed upon VRTOG (1.0) classification [8]. The steroid prescription was tapered off over six weeks. Six months after finishing radiation, the dog had a complete recheck with the neurology service. The owner reported the dog was doing well, with a stabilized appetite. Mild tremors of the legs were persistent. The neurological exam was unremarkable. The referring veterinarian performed repeat blood work before the recheck with neurology, and hematology results were within normal limits. The cholesterol and amylase values were normal, and the liver enzymes improved in comparison to the first visit but were still mildly elevated GGT 20u/l (RI 0-11u/l); ALKP 343 u/l (RI 23–212 u/l), ALT 125u/l RI(10-125U/l). Following the neurology consultation, the patient had an MRI under general anesthesia. The radiologist concluded that the size of the pituitary microadenoma remained stable, and the size of the intracranial meningioma was reduced (size: length 10.5 mm x height 12 mm, width 6 mm. The owner declined further rechecks, but regular phone calls ensured the patient's health for over two years. Three years after the initial neurology consultation, the patient was brought to the emergency service for general fatigue, locomotion incapacity, and progressive decline in quality of life. The owner elected compassionate euthanasia and allowed necropsy.
Necropsy.
On gross exam, a frontal mass was present in the right cerebral hemisphere measuring (size: length 17mm x height 12 mm, width 10 mm). The pituitary mass (length 7 mm x height 6. mm, width 5mm) was also observed grossly. Multiple adhesions were present at the level of the frontal mass. On histopathology, moderate adrenal cortical nodular hyperplasia and multifocal hemosiderosis were visualized. The liver had mild chronic centro- lobular and periportal fibrosis. The frontal mass consisted only of fibrosis and multifocal microgliosis; no neoplastic cell population was observed. Similarly, no changes were found within the pituitary gland; no microadenoma could be found.
Genetic Analysis.
Germline mutations in the menin 1 ( MEN1) gene are known to lead to "multiple endocrine neoplasia type 1" (MEN1) syndrome in people [9–13], which is characterized by the development of multifocal neoplastic endocrine lesions together with other non-endocrine tumors, such as central nervous system meningiomas [9, 14–16]. Genetic analysis was carried out to explore this patient's MEN1 gene for germline (constitutional) mutations.
After obtaining informed, written consent for participation from the owner, an EDTA whole blood sample was obtained from the patient for genetic analysis (Purdue University IACUC #1901001840). DNA was extracted via a standard phenol-chloroform extraction method and then subjected to whole-genome sequencing (WGS) using Illumina HiSeq 150bp paired-end reads, with an average of 21X coverage. Raw data quality control and variant calling were performed utilizing a previously described standardized bioinformatics pipeline [17]. The sequence reads were trimmed using Trimmomatic [18] and aligned to the canine reference genome assembly CanFam4[19] using the Burrows-Wheeler Aligner[20]. Variant calling was performed using GATK's HaplotypeCaller[21], identifying single nucleotide polymorphisms (SNPs) and indels. BCFTools 1.17[22] was used to identify private variants in the patient dog compared to WGS from seven hundred thirty -control dog genomes from genetically diverse breeds, assuming no other dog in the WGS population carried the same disease allele. Private variants were analyzed assuming both an autosomal recessive and autosomal dominant mode of inheritance, and functional effects of variants were predicted with Ensembl Variant Effect Predictor [23]. Variants predicted to have a high or medium impact on the resulting amino acid sequence were prioritized for further investigation. Each medium or high-impact variant-containing gene was filtered through VarElect[24], which prioritized genes based on how often the provided phenotypic terms "meningioma" and "pituitary tumor" appeared in conjunction with that specific gene in other gene-centric databases. Two hundred sixty-five heterozygous variants of high or moderate effect were identified under the assumption of dominant inheritance, but no apparent candidate genes previously associated with MEN1 were identified. Only three variants of high or moderate impact were identified under an assumption of recessive inheritance, and again, no evident candidate genes previously associated with MEN1 were identified. Finally, the integrative genomics viewer software[25] was used for manual visual inspection of the entire candidate gene MEN1; this failed to identify any more significant, structural private variants in the affected dog.