KLH-Based Multimodal Management of Clinically Diagnosed Canine Urothelial Carcinoma: A Two-Case Series

1. Introduction

Urothelial carcinoma (UC), historically termed transitional cell carcinoma, is the most common malignant tumor of the canine urinary bladder and may also involve the urethra, prostate, or ureters [1,2,3,4]. Canine UC frequently arises in the trigone, bladder neck, or proximal urethra, where complete surgical excision is often not feasible. Clinical consequences include hematuria, pollakiuria, dysuria, urethral or ureteral obstruction, local invasion, and metastatic spread [1,2,3,4].

Medical management therefore plays a central role. NSAIDs with cyclooxygenase-inhibitory activity have documented antitumor activity in canine UC and are frequently used alone or in combination with agents such as mitoxantrone, vinblastine, carboplatin, or doxorubicin [3,5,6,7,8,9]. Although combination protocols can achieve meaningful tumor control, treatment selection in practice is influenced by age, concurrent disease, anticipated toxicities, cost, logistical burden, and owner preference.

Detection of the canine BRAF V595E mutation in urine or tissue provides strong molecular support for UC, but a negative result does not exclude the diagnosis because a proportion of canine UCs lack detectable BRAF V595E [10,11,12]. Urine cytology, bladder tumor antigen assays, ultrasonography, CT, and MRI may contribute to a clinical diagnosis, but none replaces histopathology in all cases. False-positive bladder tumor antigen results can occur with hematuria or inflammatory urinary tract disease, and low-cellularity cytology may be nondiagnostic.

Keyhole limpet hemocyanin (KLH) is a highly immunogenic molluscan protein capable of inducing humoral and cellular immune responses. KLH-based preparations have been evaluated in human non-muscle-invasive bladder cancer, usually as intravesical or adjuvant immunotherapy after tumor resection [13,14,15,16]. Those settings differ substantially from subcutaneous KLH administration in dogs with naturally occurring invasive lower urinary tract disease. Published clinical evidence supporting this approach in canine UC is extremely limited.

This report describes the diagnostic basis, treatment protocol, longitudinal imaging findings, and apparent tolerability in two dogs managed with an NSAID, subcutaneous KLH-based immunotherapy, and selenium supplementation after cytotoxic chemotherapy was declined. The aim is to document clinical observations, not to establish causality, comparative benefit, or efficacy of any individual component.

2. Case Presentation

2.1. Treatment Rationale and Protocol

Both dogs were client-owned patients treated during routine clinical care. Standard options, including NSAID monotherapy and NSAID-based cytotoxic chemotherapy, were discussed with the owners together with anticipated benefits, adverse effects, monitoring requirements, and treatment burden. The owners declined cytotoxic chemotherapy. Following informed consent, an individualized multimodal protocol was selected with the palliative goals of preserving quality of life and limiting clinically apparent progression.

Immucothel® (immunocyanin, 1 mg; one vial) was administered subcutaneously in the right inguinal region, adjacent to the inguinal lymph node. The intended induction interval was every 2 weeks; after an acceptable clinical course, treatment intervals were extended to approximately every 4 weeks, with later adjustments in Case 2. No routine premedication was administered. Injectable selenium (Selenase® T pro injection; 200 µg/kg IV) was diluted 1:1 with 0.9% saline and administered over approximately 40 min. Patients received isotonic saline during day hospitalization. Oral selenium was omitted on injectable-selenium days. No cytotoxic anticancer drug was administered.

2.2. Case 1

An 11-year-7-month-old castrated male Maltese, born on 11 July 2011 and weighing approximately 3.15 kg at presentation on 22 February 2023, was evaluated for persistent thickening of the bladder neck and proximal urethra. A veterinary bladder tumor antigen (V-BTA) test performed on 30 September 2022 was positive. The laboratory advised interpretation with imaging and clinical findings.

Urine cytology from a low-cellularity bladder specimen identified urothelial proliferation with dysplastic features, including moderate anisocytosis and anisokaryosis, occasional binucleation, and up to two nucleoli. The cytologic interpretation was suspicious but not definitive for malignancy, and chronic inflammatory stimulation remained a differential diagnosis. CADET® BRAF and CADET® BRAF-PLUS assays did not detect the BRAF V595E mutation or additional assay targets. Histopathologic confirmation was not obtained.

Contrast-enhanced CT on 22 February 2023 showed mural thickening extending from the bladder neck into the proximal urethra. The abnormal segment was approximately 2 cm long and measured approximately 4.3 mm in maximal thickness on the contemporaneous report, with mucosal enhancement during the arterial and venous phases and more homogeneous delayed enhancement. No abdominal lymphadenopathy, pelvic invasion, or pulmonary metastatic nodule was identified. Incidental findings included gallbladder sludge, bilateral renal cortical cysts, a 5.5-mm left renal pelvic calculus, and mild left adrenal enlargement. Ultrasonography on the same date measured the proximal urethral lesion at approximately 6.75 mm. The difference between CT and ultrasonographic measurements was considered potentially attributable to imaging plane, modality, bladder distension, and lesion-margin definition (Figure 1).

Piroxicam was initiated on 22 February 2023 at 0.3 mg/kg PO q24h and continued through December 2024. Misoprostol was administered concurrently for gastrointestinal protection according to the clinical record. The KLH/selenium protocol was given as six induction treatments at approximately 2-week intervals followed by three monthly booster treatments. For Case 1, oral selenium was prescribed as Selenase® peroral solution, 500 µg PO q24h between injectable treatments; oral administration was withheld on injectable-selenium days (Figure 2).

Serial imaging showed persistent localized disease without detectable metastasis. Ultrasonographic thickness was approximately 6.9 mm in May and July 2023. Follow-up CT in August 2023 showed similar enhancement and a maximal thickness of approximately 6.4 mm, without thoracic metastasis or regional lymph-node enlargement. Ultrasonography in January 2024 measured approximately 7.5 mm; the remaining bladder wall and sublumbar lymph nodes were unremarkable.

CT on 4 August 2024 showed persistent, homogeneously enhancing thickening of the bladder neck and proximal urethra. Multiplanar CT measurements ranged from 4.05 to 4.09 mm (approximately 4.1 mm). No osseous invasion, adjacent pelvic extension, or regional lymph-node enlargement was detected. Ultrasonography on the same day measured approximately 8.1 mm, compared with 7.5 mm previously. Because CT and ultrasonographic measurements were obtained using different imaging modalities and planes, and standardized volumetric response criteria were not applied, these values were recorded descriptively rather than classified as objective progression (Figure 3).

After discontinuation of piroxicam in December 2024, clinical and imaging surveillance continued. At the latest available ultrasonographic examination, proximal urethral thickening remained approximately 6–7 mm without appreciable interval progression or regional lymphadenopathy. No urinary obstruction, detectable distant metastasis, or rescue local intervention was documented during the available follow-up.

Serial hematologic and biochemical monitoring did not identify myelosuppression or a progressive renal or hepatic abnormality that required permanent discontinuation of the KLH/selenium protocol. No immediate hypersensitivity reaction was documented. Because adverse events were not graded prospectively using VCOG-CTCAE criteria, formal safety conclusions cannot be drawn.

Given the absence of histopathology, negative BRAF assays, and non-definitive cytology, the final classification was presumptive lower urinary tract UC rather than histologically confirmed UC.

2.3. Case 2

An 11-year-old neutered male Poodle was referred after identification of a lesion at the urethrovesical junction. The dog had been receiving firocoxib (5 mg/kg PO q24h) and had not received cytotoxic chemotherapy. Baseline hematologic, biochemical, and blood-gas results were within the respective reference intervals, including hematocrit 51.8%, white blood cell count 5.24 × 10³/µL, creatinine 1.0 mg/dL, and blood urea nitrogen 24.4 mg/dL.

Contrast-enhanced CT identified an ill-defined, enhancing lesion at the urethrovesical junction measuring approximately 5 mm, while ultrasonography measured 9.0 × 6.9 mm. No regional or distant metastasis was detected at diagnosis (Figure 4). Cytologic examination had been performed at the referring hospital before presentation; however, the original report and slides were unavailable for review. A urine BRAF assay performed by IDEXX Laboratories detected the canine BRAF V595E mutation. Histopathologic confirmation was not obtained; accordingly, the case was classified as BRAF V595E-positive clinical UC on the basis of the molecular and imaging findings.

The dog continued firocoxib and received Immucothel® 1 mg SC near the inguinal lymph node, followed by intravenous selenium as described above. Oral selenium was administered as 100 µg PO q12h for 13 days between treatment visits. The first four immunotherapy cycles were administered at approximately 2-week intervals, with extension to approximately 4-week intervals beginning with the fifth cycle. Booster treatments were subsequently adjusted according to the clinical course.

Serial ultrasonography was used for follow-up because repeat CT was declined. Through the third cycle, the lesion remained approximately 10.0 × 7.0 mm. Following temporary discontinuation of firocoxib, the measured dimensions remained similar for approximately 2 weeks but increased to 11.3 × 8.7 mm by approximately 6 weeks; firocoxib was then restarted. After the sixth cycle, booster treatment was continued at approximately 4-week intervals, and a decrease to 7.4 × 5.5 mm was recorded by the eighth cycle.

When the booster interval was subsequently extended to approximately 8 weeks, the lesion was measured at 12.0 × 9.7 mm. After return to a 4-week interval, measurements decreased to 6.2 × 4.4 mm and later fluctuated around 7.6 × 4.9 mm, with approximately 4-mm residual bladder-neck thickening described on later examinations. These temporal associations cannot establish treatment responsiveness because firocoxib use, immunotherapy interval, bladder distension, imaging plane, and natural disease variation changed concurrently.

At the final follow-up on 1 July 2026, the dog had completed 18 KLH-based treatment cycles and remained alive with an acceptable clinical status after approximately 2 years of follow-up. No clinically relevant regional lymph-node enlargement, urinary obstruction, or distant metastasis was documented. No hematologic, renal, hepatic, or hypersensitivity event required permanent discontinuation; however, toxicity monitoring was retrospective and not standardized.

2.4. Comparative Clinical Summary

Table 1. Comparative summary of the two dogs. UC, urothelial carcinoma; V-BTA, veterinary bladder tumor antigen; CT, computed tomography; US, ultrasonography.

Variable	Case 1	Case 2
Signalment	Castrated male Maltese, 11 years 7 months at presentation (born 11 July 2011)	Neutered male Poodle, 11 years
Primary site	Bladder neck and proximal urethra	Urethrovesical junction
Diagnostic classification	Presumptive UC	BRAF V595E-positive clinical UC
Histopathology	Not obtained	Not obtained
BRAF V595E	Not detected	Detected
Other diagnostic support	V-BTA positive; dysplastic urothelial proliferation; serial CT/US	BRAF V595E detected (IDEXX Laboratories); CT and US; external cytology performed, but original report and slides unavailable
NSAID	Piroxicam 0.3 mg/kg PO q24h, Feb 2023–Dec 2024	Firocoxib 5 mg/kg PO q24h, with temporary interruption
KLH-based treatment	1 mg SC; 6 induction + 3 monthly boosters	1 mg SC; 18 cycles with interval adjustments
Selenium	200 µg/kg IV; 500 µg PO q24h between visits	200 µg/kg IV; 100 µg PO q12h for 13 days
Follow-up	Feb 2023–latest available follow-up	Approximately 2 years; final follow-up 1 July 2026
Metastasis detected	No	No
Interpretation	Persistent localized imaging abnormality without substantial progression	Fluctuating US measurements without documented metastatic progression

Table 1. Comparative summary of the two dogs. UC, urothelial carcinoma; V-BTA, veterinary bladder tumor antigen; CT, computed tomography; US, ultrasonography.

Variable	Case 1	Case 2
Signalment	Castrated male Maltese, 11 years 7 months at presentation (born 11 July 2011)	Neutered male Poodle, 11 years
Primary site	Bladder neck and proximal urethra	Urethrovesical junction
Diagnostic classification	Presumptive UC	BRAF V595E-positive clinical UC
Histopathology	Not obtained	Not obtained
BRAF V595E	Not detected	Detected
Other diagnostic support	V-BTA positive; dysplastic urothelial proliferation; serial CT/US	BRAF V595E detected (IDEXX Laboratories); CT and US; external cytology performed, but original report and slides unavailable
NSAID	Piroxicam 0.3 mg/kg PO q24h, Feb 2023–Dec 2024	Firocoxib 5 mg/kg PO q24h, with temporary interruption
KLH-based treatment	1 mg SC; 6 induction + 3 monthly boosters	1 mg SC; 18 cycles with interval adjustments
Selenium	200 µg/kg IV; 500 µg PO q24h between visits	200 µg/kg IV; 100 µg PO q12h for 13 days
Follow-up	Feb 2023–latest available follow-up	Approximately 2 years; final follow-up 1 July 2026
Metastasis detected	No	No
Interpretation	Persistent localized imaging abnormality without substantial progression	Fluctuating US measurements without documented metastatic progression

3. Discussion

This report describes prolonged clinical and imaging follow-up in two dogs with clinically diagnosed lower urinary tract UC managed with NSAIDs, subcutaneous KLH-based immunotherapy, and selenium supplementation. The principal contribution is descriptive documentation of diagnostic uncertainty, treatment delivery, longitudinal imaging, and tolerability when owners declined cytotoxic chemotherapy. The observations do not demonstrate that Immucothel®, selenium, or the combined regimen produced tumor control.

The diagnostic evidence differed substantially between cases. Case 2 had BRAF V595E detection together with compatible imaging, providing strong molecular support for UC; although cytology had been performed at the referring hospital, the original report and slides were unavailable for review. Case 1 had a positive V-BTA result, dysplastic urothelial proliferation on low-cellularity cytology, and persistent characteristic lower urinary tract thickening, but both BRAF assays were negative and histopathology was unavailable. Approximately 15–20% of canine UCs may lack detectable BRAF V595E, so a negative assay does not exclude UC [10,11,12]. Nevertheless, the non-definitive cytology and potential for false-positive V-BTA results require continued use of the qualifier 'presumptive' for Case 1.

NSAIDs are a major confounder. Piroxicam and firocoxib can exert antitumor activity in canine UC, and NSAID monotherapy or NSAID-containing protocols have produced objective responses and disease stabilization in prior studies [5,6,7,8,9]. Both dogs received long-term NSAIDs during much of the observation period. In Case 2, enlargement was recorded after temporary firocoxib withdrawal and stabilization after reintroduction, further preventing attribution of the course to KLH-based immunotherapy.

KLH is highly immunogenic, but the biological relevance of the protocol used here remains unproven. Human studies evaluated KLH chiefly for recurrence prevention in non-muscle-invasive bladder cancer, commonly after transurethral resection and by intravesical or other schedules that differ from the subcutaneous protocol reported here [13,14,15,16]. Neither anti-KLH antibody titers, anti–Thomsen–Friedenreich antibodies, lymphocyte responses, cytokines, natural-killer-cell activity, nor tumor-associated immune markers were measured. Thus, the report cannot confirm that a biologically meaningful systemic immune response occurred.

The role of selenium is also indeterminate. Selenium participates in redox regulation and immune function, and experimental anticancer effects have been described [17,18]. However, selenium has a narrow margin between nutritional adequacy and toxicity, and clinical antitumor benefit has not been established for this dose and schedule in canine UC. No serum selenium concentration, selenoprotein activity, or pharmacodynamic marker was measured. Selenium therefore represents an additional uncontrolled intervention.

Serial imaging suggested a prolonged absence of substantial local progression and detectable metastasis, particularly in Case 1. However, measurements obtained by CT and ultrasonography are not directly interchangeable. Bladder distension, imaging plane, operator technique, mural-versus-luminal margins, and lesion geometry can alter measured thickness. Because prospective RECIST-like or canine bladder tumor response criteria were not applied, formal terms such as complete response, partial response, stable disease, and progressive disease were intentionally avoided.

The absence of detected metastasis is clinically noteworthy but cannot be interpreted as prevention of metastasis. The clinical course and reasons for euthanasia in canine UC are heterogeneous [20], and patients with more indolent localized disease are more likely to remain available for prolonged follow-up, creating selection and survivorship bias. Furthermore, staging frequency and modality varied over time, particularly in Case 2, for which follow-up relied predominantly on ultrasonography.

Apparent tolerability was acceptable in these two dogs, but safety cannot be established from a retrospective two-case series. Concurrent fluids and gastrointestinal protection may have influenced tolerability. Adverse events were not prospectively graded according to Veterinary Cooperative Oncology Group–Common Terminology Criteria for Adverse Events (VCOG-CTCAE v2), and uncommon or delayed adverse effects cannot be excluded [19].

Despite these limitations, the cases may be useful as hypothesis-generating observations. A future study should prospectively require histopathology or robust predefined multimodal diagnostic criteria; stratify by BRAF status, disease stage, and tumor site; standardize the NSAID, KLH, and selenium regimens; include an NSAID-only comparator; apply objective imaging and quality-of-life endpoints; use formal adverse-event grading; and measure anti-KLH antibodies and cellular immune biomarkers. Such a design would be necessary to determine whether subcutaneous KLH has biological activity beyond established NSAID effects.

4. Limitations

The major limitations are the retrospective uncontrolled design; inclusion of only two dogs; absence of histopathologic confirmation in both cases; weaker diagnostic certainty in Case 1; concurrent NSAID and selenium administration; owner- and clinician-directed changes in treatment intervals; nonstandardized imaging and response assessment; lack of formal toxicity grading; absence of immunologic monitoring; incomplete control of concomitant medications; and risks of selection, survivorship, and publication bias. These limitations preclude conclusions regarding efficacy, comparative benefit, metastatic prevention, or the independent contribution of KLH-based immunotherapy.

5. Conclusions

Two dogs with clinically diagnosed lower urinary tract UC showed prolonged clinical stability and no documented metastatic progression while receiving NSAIDs, subcutaneous KLH-based immunotherapy, and selenium supplementation after cytotoxic chemotherapy was declined. The regimen was not associated with an adverse event requiring permanent discontinuation in these individual patients. However, the multimodal uncontrolled design, lack of histopathology, nonstandardized response assessment, and absence of immune monitoring prevent attribution of the observed courses to KLH-based immunotherapy. The report should be regarded as descriptive and hypothesis-generating. Prospective controlled investigation is required before this approach can be recommended as an alternative to established treatment.