• View in gallery
    Figure 1—

    Scintigraphic images of a clinically normal dog (A) and a dog with histologically confirmed B-cell lymphoma (B) in right lateral recumbency 1 hour after IV administration of the RaPP 111In-DOTA-anti–BCL2–PNA-Tyr3-octreotate. In panel A, notice concentrations of the radiolabeled RaPP in regions of the heart and urinary bladder of the healthy dog. In panel B, notice multiple regions of increased intensity indicative of RaPP uptake. Images were colorized by use of image analysis software. Intensity is indicated by color as follows: blue, green, yellow, and red, where blue represents the lowest value and red represents the highest value. B = Bladder. K = Kidney. KS = Kidney (shielded). LS = Lead shield. M = Mandibular lymph node. P = Popliteal lymph node. SC = Superficial cervical lymph node.

  • View in gallery
    Figure 2—

    Box-and-whiskers plot showing results of subjective interpretation of ROI intensity in scintigraphic images of 11 dogs with B-cell lymphoma after IV RaPP administration. Upper and lower limits of the boxes represent the interquartile range between the 25th and 75th percentiles; horizontal lines represent the median value. Whiskers extend to the minimum and maximum data points unless values were ≥ 1.5 times the interquartile range; these are represented by solid circles. Regions of interest included mandibular, superficial cervical, and popliteal lymph nodes; liver; and spleen. The intensity of each ROI (expressed as a ratio relative to the intensity of an equally sized region of muscle in the same image) was measured by use of computer software. Each ROI was also subjectively categorized as positive (n = 22) or negative (74) for increased RaPP uptake by 1 investigator (JCL), and the median values of objectively measured intensities for the 2 groups were compared. The median value of positive ROIs (1.9) was significantly (P < 0.001) greater than that of negative ROIs (1.3).

  • View in gallery
    Figure 3—

    Dot plot of TTP in 10 dogs with B-cell lymphoma grouped according to subjective categorization of the superficial cervical lymph node ROI as negative (n = 8) or positive (2) for increased RaPP uptake. One dog that was euthanized because of squamous cell carcinoma (while still in durable remission for B-cell lymphoma) was not included in the analysis. Dogs for which the ROI was considered negative had a significantly (P = 0.044) longer TTP (median, 268.5 days; range, 230 to 522 days) than did those for which it was considered positive (median, 126.0 days; range, 78 to 174 days).

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Evaluation of a B-cell leukemia-lymphoma 2-specific radiolabeled peptide nucleic acid–peptide conjugate for scintigraphic detection of neoplastic lymphocytes in dogs with B-cell lymphoma

Kimberly A. Statham-RingenDepartment of Veterinary Medicine and Surgery, College of Veterinary Medicine, University of Missouri, Columbia, MO 65211.

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Kimberly A. SeltingDepartment of Veterinary Medicine and Surgery, College of Veterinary Medicine, University of Missouri, Columbia, MO 65211.

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Jimmy C. LattimerDepartment of Veterinary Medicine and Surgery, College of Veterinary Medicine, University of Missouri, Columbia, MO 65211.

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Carolyn J. HenryDepartment of Veterinary Medicine and Surgery, College of Veterinary Medicine, University of Missouri, Columbia, MO 65211.
Division of Hematology and Oncology, Department of Internal Medicine, School of Medicine, University of Missouri, Columbia, MO 65211.

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Jonathan A. GreenDivision of Animal Sciences, College of Agriculture, Food and Natural Resources, University of Missouri, Columbia, MO 65211.

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Jeffrey N. BryanDepartment of Veterinary Clinical Sciences, College of Veterinary Medicine, Washington State University, Pullman, WA 99164.

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Fang JiaDepartment of Veterinary Medicine and Surgery, College of Veterinary Medicine, University of Missouri, Columbia, MO 65211.
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Michael R. LewisDepartment of Veterinary Medicine and Surgery, College of Veterinary Medicine, University of Missouri, Columbia, MO 65211.
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Abstract

Objective—To evaluate use of a radiolabeled peptide nucleic acid–peptide conjugate (RaPP) targeting B-cell leukemia-lymphoma 2 (BCL2) mRNA for scintigraphic detection of neoplastic lymphocytes in dogs with B-cell lymphoma and to assess associations among RaPP uptake, time to tumor progression (TTP), and BCL2 mRNA expression.

Animals—11 dogs with B-cell lymphoma and 1 clinically normal dog.

Procedures—Scintigraphic images were acquired 1 hour after IV injection of the RaPP. Regions of interest (ROIs) were drawn around lymph nodes, liver, and spleen; ROI intensity (relative to that of an equally sized region of muscle in the same image) was measured. Each ROI was also subjectively categorized as positive or negative for increased RaPP uptake. Expression of BCL2 mRNA was determined via quantitative reverse transcriptase PCR assay of a lymph node sample from dogs with lymphoma. Associations among imaging results, TTP, and BCL2 mRNA expression were evaluated.

Results—Increased RaPP uptake was detected in affected tissues of dogs with lymphoma. Dogs with superficial cervical lymph node ROIs categorized as negative (n = 8) for increased RaPP uptake had a significantly longer TTP than did dogs for which this ROI was considered positive (2). Measured intensity of mandibular and superficial cervical lymph node ROIs was negatively associated with TTP. Associations among BCL2 mRNA and ROI intensity or TTP were not significant.

Conclusions and Clinical Relevance—Increased RaPP uptake at mandibular or superficial cervical lymph node ROIs may be a negative prognostic indicator in dogs with lymphoma. A larger investigation is needed to determine clinical value of the RaPP for disease detection and prognostication.

Abstract

Objective—To evaluate use of a radiolabeled peptide nucleic acid–peptide conjugate (RaPP) targeting B-cell leukemia-lymphoma 2 (BCL2) mRNA for scintigraphic detection of neoplastic lymphocytes in dogs with B-cell lymphoma and to assess associations among RaPP uptake, time to tumor progression (TTP), and BCL2 mRNA expression.

Animals—11 dogs with B-cell lymphoma and 1 clinically normal dog.

Procedures—Scintigraphic images were acquired 1 hour after IV injection of the RaPP. Regions of interest (ROIs) were drawn around lymph nodes, liver, and spleen; ROI intensity (relative to that of an equally sized region of muscle in the same image) was measured. Each ROI was also subjectively categorized as positive or negative for increased RaPP uptake. Expression of BCL2 mRNA was determined via quantitative reverse transcriptase PCR assay of a lymph node sample from dogs with lymphoma. Associations among imaging results, TTP, and BCL2 mRNA expression were evaluated.

Results—Increased RaPP uptake was detected in affected tissues of dogs with lymphoma. Dogs with superficial cervical lymph node ROIs categorized as negative (n = 8) for increased RaPP uptake had a significantly longer TTP than did dogs for which this ROI was considered positive (2). Measured intensity of mandibular and superficial cervical lymph node ROIs was negatively associated with TTP. Associations among BCL2 mRNA and ROI intensity or TTP were not significant.

Conclusions and Clinical Relevance—Increased RaPP uptake at mandibular or superficial cervical lymph node ROIs may be a negative prognostic indicator in dogs with lymphoma. A larger investigation is needed to determine clinical value of the RaPP for disease detection and prognostication.

The BCL2 gene is a member of a family of genes that regulate apoptosis. Overexpression of BCL2 protein has been associated with increased relapse rates, shorter TTP, and decreased survival times in human patients with non-Hodgkin lymphoma.1–3 Three-year survival rates of 30% and 70% have been reported for human patients with CLL and follicular forms of non-Hodgkin lymphoma that have high or low BCL2 expression in biopsy samples, respectively2–4 As an antiapoptotic protein, BCL2 can contribute to resistance to chemotherapeutic agents and radiation.5–7

Peptide receptor targeting for diagnosis and treatment of cancers has become a focus of nuclear medicine in humans. However, these technologies are rarely used in veterinary patients. Peptide-based agents can target cells in vivo by agonist-induced receptor internalization and intracellular retention of the radiolabeled agent in tumor cells.8,9 A peptide receptor commonly used for in vivo cancer targeting is the somatostatin receptor, which can be highly expressed in neuroendocrine tumors and have lower expression levels in lymphoproliferative disorders, including lymphoma.10–12

Lymphoma is a common neoplastic disease in dogs and was reported to affect 1.2% of all dogs in a large retrospective study13 that included records from veterinary teaching hospitals spanning a 38-year period. Current treatment recommendations consist of multidrug protocols that result in 60% to 90% complete remission rates and median survival times ranging from 6 to 12 months.14–16 Unfortunately, reported response rates to conventional chemotherapy in canine patients have not changed in > 20 years. Several prognostic indicators have been identified for lymphoma in dogs, including immunophenotype, World Health Organization substage, and mediastinal involvement.17–19 Results of 1 study20 in 23 dogs with multicentric lymphoma indicated that BCL2 expression was not correlated with resistance to chemotherapeutic agents; however, TTP and other outcome measures were not evaluated. Results of experimental research in rodents as well as human cancer research have indicated that BCL2 has a role in drug resistance, suggesting that further investigation is warranted.2–5

Noninvasive imaging of BCL2 expression can potentially be used to identify high-risk patients with lymphoma that may be resistant to conventional chemotherapeutic agents. Molecular imaging of BCL2 expression has been described in immunodeficient mice engrafted with Mec-1 (human B-cell CLL) cells by investigators who used an RaPP to target BCL2 gene expression.21 In that study,21 the radiolabeled PNA-somatostatin analogue conjugate, γ-emitting 111In-DOTA-anti–BCL2–PNA-Tyr3-octreotate, was confirmed to be internalized and to have specific receptor-mediated cell uptake and targeting of human BCL2 mRNA.

Favorable biodistribution and tumor-specific uptake of the somatostatin analogue, 111In-Tyr3-octreotate, has been demonstrated in a variety of tumor tissues in rats and mice. In a study22 performed to evaluate tumor-specific uptake of various 111In-labeled somatostatin analogues, Tyr3-octreotate had the highest uptake in somatostatin receptor–positive tumors and the greatest degree of binding and internalization by tumor cells in vivo, compared with other compounds tested.

Importantly, the radionuclide coupled to the conjugate is internalized by the targeted cell after binding to the somatostatin receptor. The internalization of somatostatin analogues occurs via G-protein coupling, and peptide analogues are targeted for degradation during receptor recycling.23 In contrast, PNA analogues are resistant to peptidases and remain highly stable in biological systems. Specificity of the RaPP binding to BCL2 mRNA within the cell is accomplished via the PNA, which is a DNA-like antisense molecule that directs the conjugate to the mRNA of interest.

The purpose of the study reported here was to determine whether whole-body nuclear scintigraphy could be used to detect neoplastic lymphocytes in dogs with histologically confirmed B-cell lymphoma following IV administration of the RaPP 111In-DOTA-anti–BCL2–PNA-Tyr3-octreotate and to assess associations among RaPP uptake, TTP, and BCL2 mRNA expression in an affected lymph node. We hypothesized that dogs with naturally occurring B-cell lymphoma would have specific uptake of the RaPP in neoplastic tissues and that increased specific uptake of the RaPP in predetermined ROIs would be directly correlated with BCL2 mRNA expression and negatively associated with TTP.

Materials and Methods

Animals—Eleven dogs admitted to the University of Missouri Veterinary Medical Teaching Hospital for evaluation and treatment of B-cell lymphoma were prospectively enrolled in the study between July 2007 and October 2009. One healthy privately owned dog was included in the study for imaging comparison purposes. The previous medical history and lack of clinical signs as well as results of a physical examination were used to determine health status of this dog. Disease stage in all dogs with lymphoma was assessed on the basis of results of a CBC, serum biochemical analysis including electrolytes, urinalysis, thoracic and abdominal radiography, abdominal ultrasonographic examination, and bone marrow aspiration with core biopsy. Abdominal organs were considered to have lymphoma involvement if the ultrasonographic examination revealed enlargement, echogenicity changes, or both consistent with those previously reported as typical of visceral lymphoma.24–26 Ultrasound-guided fine-needle aspirates were obtained to confirm lymphoma infiltrate of hepatic and splenic parenchyma if the echogenicity was considered normal or if echogenicity was not consistent with previously reported typical visceral lymphoma.

Dogs with a mediastinal mass or hypercalcemia were excluded in an effort to avoid enrollment of dogs with T-cell lymphoma prior to definitive diagnosis. Dogs that had received chemotherapeutic drugs prior to the start of the study (other than prednisone, which was allowed if discontinued ≥ 2 weeks prior to enrollment) or had evidence of hepatic or renal deficiency as defined by serum total bilirubin concentration ≥ 0.6 mg/dL (reference range, 0.1 to 0.6 mg/dL), alanine aminotransferase activity ≥ 232 U/L (reference range, 9 to 58 U/L), and creatinine concentration ≥ 1.8 mg/dL were also excluded. A diagnosis of histologically confirmed B-cell lymphoma was required for enrollment in the study. Histologic confirmation consisted of microscopic evaluation of an excisional lymph node biopsy specimen obtained prior to initial scintigraphic imaging, with detection of the CD79a antigen via immunohistochemistry performed by a board-certified pathologist at the University of Missouri Veterinary Medical Diagnostic Laboratory. The study protocol was reviewed and approved by the University of Missouri Animal Care and Use Committee, and owner consent was obtained for privately owned dogs prior to study enrollment.

All dogs with lymphoma underwent the same previously described University of Wisconsin-Madison chemotherapy protocol (ie, UW-25) of l-asparaginase, cyclophosphamide, doxorubicin, vincristine, and prednisone.27 Clinical outcome was assessed via evaluation of the TTP, which was defined as the number of days between initiation of chemotherapy (day 1) and detectable relapse of disease. Durable remission was defined as the lack of detectable disease as determined on the basis of physical examination performed weekly from weeks 1 through 8 as well as results of the following: CBC, serum biochemical analysis, thoracic and abdominal radiography, and abdominal ultrasonography between weeks 6 and 8 of the UW-25 protocol. Cytologic evaluation of palpably normal-sized lymph nodes was not performed. Relapse was confirmed if cytologic evaluation of a fine-needle aspirate from a palpably enlarged lymph node that had reduced in size during chemotherapy revealed a predominant population of intermediate to large lymphocytes consistent with lymphoma. Patients were censored if they developed an unrelated comorbidity. Survival time was not evaluated because treatments administered at the time of disease relapse varied among patients.

Scintigraphic imaging—The RaPP 111In-DOTA-anti–BCL2–PNA-Tyr3-octreotate was prepared prior to administration as described previously.21 The PNA sequence, complementary to the first 14 bases of the open reading frame of the BCL2 gene, was identical for dogs (GenBank accession No. AB154172.1) and humans (GenBank accession No. NM_000633.2). In addition, the canine somatostatin receptor type 2 sequence (GenBank accession No. NP_001026987.1) was identical to the corresponding sequence in humans (GenBank accession No. NP_001041.1). Eleven dogs with B-cell lymphoma and 1 clinically normal dog each received 1 dose of 74 to 111 MBq (2 to 3 mCi) of the RaPP through an aseptically placed peripheral indwelling IV catheter in either the lateral saphenous or cephalic vein after sedation. The dose range resulted from small variations in the amount of radiopharmaceutical available at the time of imaging.

Scintigraphic images were obtained 1 hour after administration of the RaPP. Whole-body scintigraphic images were acquired by use of a planar gamma cameraa equipped with a medium energy all-purpose collimator. To enable direct comparisons, images were count-rate matched at the time of acquisition. All dogs were sedated with morphine (0.5 mg/kg, IV, once), glycopyrrolate (0.005 mg/kg, IV, once), and medetomidine HCl (10 μg/kg, IV, once) for positioning and image acquisition. The urinary bladder and kidneys were shielded with lead blocks to minimize artifacts caused by accumulation of the renally excreted imaging agent in the urinary tract. Images were obtained in ventrodorsal as well as left and right lateral recumbency, and all views were acquired with an acquisition time of 60 seconds for each position. Dogs were kept in radiation isolation until 48 hours after injection, in accordance with Nuclear Regulatory Commission regulations. These regulations mandate an 111In exposure rate of ≤ 1.03 mrem/h at a distance of 1 m for release, with a minimum isolation time of 48 hours. Dogs were monitored once for radiation emission before discharge at 48 hours, at which time all met the release criteria.

Images were obtained at 3 times during the course of treatment: prior to the initiation of chemotherapy, at durable remission, and at clinical relapse of disease. Images were converted to JPG file format and imported into image analysis software.b Regions of interest were drawn around the peripheral (mandibular, superficial cervical, and popliteal) lymph nodes, liver, and spleen. The intensity of these regions, determined with computer software, was compared with that of an equally sized region of muscle in same image. The scintigrams were colorized by use of image analysis software.b Determining this value as a ratio of ROI intensity to the background intensity of muscle provided an internal control to account for qualitative variations in lymph node size among patients. Each ROI was then subjectively categorized as positive or negative on the basis of the professional experience of the radiologist. All scintigraphic images were evaluated by a board-certified veterinary radiologist with experience in nuclear medicine imaging (JCL) who was blinded as to patient data and case outcome. Relative intensity of the excisional biopsy site ROI could not be included in statistical evaluations because the images were acquired between 24 and 48 hours after the excisional biopsy procedure. Tissues with increased RaPP uptake were confirmed to have lymphoma involvement if ultrasonographic interpretation of tissues was consistent with lymphoma or if infiltration of visceral organs or palpably enlarged lymph nodes was confirmed via cytologic evaluation.

cDNA standard preparation—An 8-point standard curve (r2 = 0.99) was generated with 1:10 serial dilutions of a canine BCL2 cDNA plasmid preparationc that had an initial concentration of 1.79 × 10−1 μg/μL. Plasmid growth, isolation, and purification were performed in accordance with the manufacturer's instructions.d The insert DNA was sequenced by the University of Missouri DNA Research Core. The insert was identical to the canine BCL2 mRNA (GenBank accession No. AB116145.1).

Tissue samples for RT-PCR assay—Histologically confirmed lymphoma tissue specimens were collected via excisional biopsy of a superficial cervical or popliteal lymph node from dogs at the time of initial staging, prior to scintigraphic imaging. All fresh tissue specimens were snap frozen immediately after collection in liquid nitrogen and stored at −80°C until analysis.

RNA isolation and first-strand synthesis—Total RNA was extracted from lymph node tissue (30 mg) with RNA reagente in accordance with the manufacturer's instructions. The resulting pellet was dissolved in 200 μL of diethylpyrocarbonate-treated water. An aliquot of 1 μL of RNase inhibitorf was then added. The RNA yield and the ratio of absorbance at 260 to 280 nm were measured by use of a spectrophotometer.g A DNase treatment was performed with RNase-free DNase, followed by an RNA cleanup protocol performed with a commercially available kit, as recommended by the manufacturer.h Integrity of the RNA was analyzed via electrophoresis on 1% agarose gel and evaluation of the ethidium bromide–stained product under UV light. Reverse transcription was performed by use of a proprietary RTi with oligo dT hexamers used as recommended by the manufacturer. The amount of RNA used in each reaction was 2 μg. The resulting first-strand cDNA was stored at −20°C and underwent only 1 freeze-thaw cycle before being used in a 2-step quantitative RT-PCR method.

Absolute quantitative minor groove binder RT-PCR assay—Quantitative RT-PCR assays for canine BCL2j were performed under the following conditions: 95°C for 10 minutes, 40 cycles of 95°C for 15 seconds, and a final step of 60°C for 1 minute. This assay was used to amplify an 85-bp product in the unknown and standard cDNA samples. The primer and probe sequences used were identical to National Center for Biotechnology Information reference sequence NM_001002949.1 and have been validated and compared with 3 GenBank sequences (accession Nos. AB154172.1, AY509563.1, and AB116145.1). The latter sequence was identical to the canine BCL2 cDNA insert used to generate the standard curve in the present study. The quantitative RT-PCR assay was conducted in a thermocyclerk in a total volume of 20 μL/reaction that contained 1 μL of template, 10 μL of universal master mix, 18μM each of forward and reverse primer, and 5μM of probe. The probe was labeled with a 6-carboxyfluorescein reporter dye,l and a 5-carboxy-X-rhodamine–based reference dyem was used to normalize the fluorescent reporter signal. Assay setup and analysis were performed as recommended by the manufacturer.k The threshold cycle was defined as the cycle at which the fluorescence was noticeably higher than the mean ± SD of the preceding cycles and the sequence detection application began to detect the increase in signal associated with an exponential growth of the PCR product. Standard samples were prepared in an identical manner to that used for the unknown reactions. Triplicate reactions were performed for unknown and standard samples to ensure accuracy of quantitation.

Quantitative RT-PCR analysis—Absolute quantitative RT-PCR assay results were analyzed by use of commercially available software.n The default settings of the program were used to define both the threshold value and baseline parameters for analysis of the raw data. Amounts of BCL2 mRNA in the unknown samples were extrapolated from the concurrently performed standard curve.

Statistical analysis—All analyses were performed with a statistical software package.o To determine whether the subjective categorization of ROIs in scintigraphic images as positive or negative for increased RaPP uptake could serve as a prognostic indicator for clinical outcome, regression models were fitted and refined; TTP was used as the outcome variable, and subjective ROI intensity categories were considered explanatory variables. To determine the role of BCL2 mRNA expression as a prognostic indicator, Cox proportional hazards regression models were fitted and refined by sequential deletion of variables, with TTP used as the outcome variable and BCL2 mRNA concentration, biopsy site, and anatomic site considered explanatory variables. Cox proportional hazards regression models were similarly used to determine the prognostic value of objectively measured relative intensity of ROIs after RaPP administration, with TTP used as the outcome variable and relative image intensities at each ROI considered explanatory variables. Separate models were developed for each time point (initial evaluation, durable remission, and relapse of clinical disease). A Kruskal-Wallis rank sum test was used to test for differences in median relative intensity between ROIs subjectively grouped as positive or negative for increased RaPP uptake. To evaluate the correlation between image intensity of the RaPP and BCL2 mRNA expression, a Pearson product-moment correlation coefficient was used. Two-sided tests were used in all aspects of the study to enable evaluation of both negative and positive relationships.

Results

The 11 dogs with B-cell lymphoma included 2 German Shepherd Dogs, 3 Labrador Retrievers, 2 mixed-breed dogs, and Scottish Terrier, Rottweiler, Beagle, and American Eskimo Dog (1 each). Of these, 7 were castrated males, 3 were spayed females, and 1 was a sexually intact female. Median weight was 35.4 kg (range, 7 to 53.5 kg) and median age was 10.2 years for these dogs; World Health Organization lymphoma stages included stage IV substage a (n = 6 dogs) and stage V substages a (4) and b (1). Lymph nodes excised for histologic confirmation of B-cell lymphoma and gene expression assays in affected dogs were popliteal (n = 9) or superficial cervical (2). One dog had previously received prednisone treatment, and the remaining affected dogs had not received any treatment for lymphoma at the time of the initial evaluation. The clinically normal dog was a castrated male mixed-breed dog that weighed 13 kg.

Durable clinical remission was achieved in all but 1 affected dog. The remaining dog had a partial clinical remission with progressive disease detected at day 78. The median TTP was 243 days (range, 78 to 522 days). Ten of the 11 dogs were included in the analyses for TTP; 1 dog was censored when it was euthanized because of squamous cell carcinoma while still in durable remission for B-cell lymphoma.

Scintigraphic imaging—Results from all 11 dogs with lymphoma and the clinically normal dog were used to evaluate relative intensity of ROIs (indicative of RaPP uptake) in scintigraphic images. No image quality differences attributable to the dose of RaPP could be subjectively identified. During evaluation of images of the clinically normal dog, increased intensity was detected in areas such as the kidneys, bladder, and cardiac region; this was attributed to excretion of the radiopharmaceutical and to vascular pooling (Figure 1). Subjective evaluation of scintigraphic images obtained during the initial evaluation of all enrolled dogs revealed multiple regions with increased RaPP uptake in tissues confirmed to have lymphoma involvement. No adverse clinical effects attributable to RaPP administration were reported during the study.

Figure 1—
Figure 1—

Scintigraphic images of a clinically normal dog (A) and a dog with histologically confirmed B-cell lymphoma (B) in right lateral recumbency 1 hour after IV administration of the RaPP 111In-DOTA-anti–BCL2–PNA-Tyr3-octreotate. In panel A, notice concentrations of the radiolabeled RaPP in regions of the heart and urinary bladder of the healthy dog. In panel B, notice multiple regions of increased intensity indicative of RaPP uptake. Images were colorized by use of image analysis software. Intensity is indicated by color as follows: blue, green, yellow, and red, where blue represents the lowest value and red represents the highest value. B = Bladder. K = Kidney. KS = Kidney (shielded). LS = Lead shield. M = Mandibular lymph node. P = Popliteal lymph node. SC = Superficial cervical lymph node.

Citation: American Journal of Veterinary Research 73, 5; 10.2460/ajvr.73.5.681

Association between subjective ROI categorization after RaPP administration and TTP—Results of analysis of scintigraphic images obtained during the initial evaluation indicated that ROIs subjectively categorized as positive for increased RaPP uptake (n = 22) were significantly (P < 0.001) higher in relative intensity (median value, 1.9) than were negative ROIs (74; median value, 1.3; Figure 2). Multiple ROIs were evaluated for each dog; in some dogs, not all ROIs could be evaluated, depending on which tissues were involved with disease. The superficial cervical lymph node ROI was the only site for which the category was significantly associated with TTP. Dogs for which this site was negative (n = 8; median TTP, 268.5 days; range, 230 to 522 days) had a significantly (P = 0.044) longer TTP than did those for which the site was positive (2; median TTP, 126.0 days; range, 78 to 174 days; Figure 3). No significant differences in TTP were detected in any additional regions at remaining time points (durable remission and relapse).

Figure 2—
Figure 2—

Box-and-whiskers plot showing results of subjective interpretation of ROI intensity in scintigraphic images of 11 dogs with B-cell lymphoma after IV RaPP administration. Upper and lower limits of the boxes represent the interquartile range between the 25th and 75th percentiles; horizontal lines represent the median value. Whiskers extend to the minimum and maximum data points unless values were ≥ 1.5 times the interquartile range; these are represented by solid circles. Regions of interest included mandibular, superficial cervical, and popliteal lymph nodes; liver; and spleen. The intensity of each ROI (expressed as a ratio relative to the intensity of an equally sized region of muscle in the same image) was measured by use of computer software. Each ROI was also subjectively categorized as positive (n = 22) or negative (74) for increased RaPP uptake by 1 investigator (JCL), and the median values of objectively measured intensities for the 2 groups were compared. The median value of positive ROIs (1.9) was significantly (P < 0.001) greater than that of negative ROIs (1.3).

Citation: American Journal of Veterinary Research 73, 5; 10.2460/ajvr.73.5.681

Figure 3—
Figure 3—

Dot plot of TTP in 10 dogs with B-cell lymphoma grouped according to subjective categorization of the superficial cervical lymph node ROI as negative (n = 8) or positive (2) for increased RaPP uptake. One dog that was euthanized because of squamous cell carcinoma (while still in durable remission for B-cell lymphoma) was not included in the analysis. Dogs for which the ROI was considered negative had a significantly (P = 0.044) longer TTP (median, 268.5 days; range, 230 to 522 days) than did those for which it was considered positive (median, 126.0 days; range, 78 to 174 days).

Citation: American Journal of Veterinary Research 73, 5; 10.2460/ajvr.73.5.681

Association between relative intensity of ROIs after RaPP administration and TTP—At the time of initial evaluation, the minimum significant model for predicting TTP by use of objectively measured relative intensity of ROIs in scintigraphic images included values for the mandibular and superficial cervical lymph node regions (n = 10 dogs; P = 0.002). This combined model performed better than models that included values for either site alone. Hazard ratios indicated that as mandibular lymph node ROI intensity increased by 1 multiple of the background intensity (with superficial cervical lymph node intensity remaining the same), the risk of tumor progression was increased approximately 44.2-fold (95% CI, 1.48 to 1,322; P = 0.029). A 1-unit increase in superficial cervical lymph node ROI intensity was associated with an approximately 144-fold increased risk of tumor progression (95% CI, 1.92 to 10,798; P = 0.024). Associations between ROI intensity of viscera (spleen and liver) or caudal regional lymph nodes and TTP were not significant. Intensity of evaluated ROIs (liver, spleen, and mandibular, superficial cervical, and popliteal lymph nodes) after RaPP administration was not significantly different between scintigraphic images obtained during initial evaluation and those obtained during remission and relapse.

Correlation between relative intensity of ROIs after RaPP administration and BCL2 mRNA expression—Results from all 11 dogs with lymphoma were used to evaluate the relationship between BCL2 mRNA expression in an affected lymph node and the relative intensity of ROIs in scintigraphic images. Quantitative RT-PCR data revealed cycle threshold values in unknown samples with a range from 21 to 27 cycle thresholds. Concentrations of BCL2 mRNA extrapolated from the standard curve had a median value of 1.55 × 10−5 μg/μL. There was very little variation in BCL2 mRNA expression among dogs of the present study. No significant correlation was revealed between BCL2 mRNA concentration in the excised lymph node sample and ROI intensity at any site at any time point. A nonsignificant (P = 0.12) positive correlation was detected between mandibular lymph node ROI intensity and BCL2 mRNA concentration (R2 = 0.49).

Association between BCL2 mRNA expression and TTP—Results from 10 dogs with lymphoma were used to evaluate the relationship between BCL2 mRNA expression in an affected lymph node and TTP. No significant (P = 0.83) association between these variables was detected.

Discussion

In the study reported here, we evaluated tissue-specific uptake of the RaPP 111In-DOTA-anti–BCL2–PNA-Tyr3-octreotate in dogs with B-cell lymphoma. These dogs underwent a previously described chemotherapeutic regimen27 that began after the initial scintigraphic evaluation and had expected rates of response to treatment and TTP. Results of subjective and objective analysis of scintigraphic images after RaPP administration revealed that increased uptake of the RaPP in the mandibular and superficial cervical lymph nodes was associated with increased risk of tumor progression.

We evaluated the relationship between scintigraphic imaging results and clinical outcome (assessed as TTP) in several ways. Limitations involved with interpretation of the scintigraphic images may have hampered our ability to detect meaningful differences in some regions. When evaluating viscera and caudal regional lymph nodes, the inability to detect significant associations could simply have been related to location and overlapping high intensities from the kidneys and urinary bladder because the imaging agent is primarily excreted through the urinary system.28 This could be minimized via placement of a urinary catheter to evacuate the bladder and thus reduce the amount of radioactive material concentrated in the caudal abdomen, allowing improved interpretation of tissue intensity in these regions. In contrast, the superficial location of the mandibular and superficial cervical lymph nodes, together with minimal surrounding blood pool and distance from excretory reservoirs, allowed improved image interpretation for all patients for these ROIs. Associations detected between RaPP uptake (as determined by relative intensity) at the superficial cervical and mandibular lymph node ROIs and TTP were encouraging because they were less affected by artifact than were viscera and caudal regional lymph nodes.

In analysis of scintigraphic images after RaPP administration during initial evaluation, ROIs subjectively categorized as positive had significantly (P < 0.001) higher intensities than did those categorized as negative. The significant (P = 0.044) association between TTP and subjective categorization (positive or negative) of the superficial cervical lymph node site supported the ability of clinicians to provide prognostic information on the basis of subjective image interpretation and suggested that dogs with positive results for increased RaPP uptake in this region could potentially have significantly shorter TTP, compared with dogs that have negative results.

There was a significant association between TTP and objectively measured relative intensities of the mandibular and superficial cervical lymph node ROIs in scintigraphic images obtained at the initial evaluation, with relative intensities for these 2 regions explaining 68.6% of the variation in TTP. These results indicated that the risk of tumor progression increased approximately 44-fold when the intensity of the mandibular lymph node ROI increased by 1 multiple of the background intensity and approximately 144-fold when intensity of the superficial cervical lymph node ROI increased by the same 1-unit increment. Although the CIs for these values were very wide, indicating a lack of precision in the hazard ratio estimates and the lower limits of the CIs were approximately the same, neither included 1.0. Therefore, we considered our results to strongly suggest that increases in relative intensity at mandibular or superficial cervical lymph node ROIs are negative prognostic indicators.

The lack of difference between ROI intensities measured after RaPP administration during the initial evaluation and those measured during remission and relapse may have been attributable to a lack of difference in BCL2 mRNA expression at these time points. Human patients in long-term remission for CLL after first-line treatment may still have detectable circulating cells positive for t(14;18), the most common chromosomal translocation associated with BCL2, without any clinical evidence of active disease.29 Therefore, minimal residual disease, below the limit of clinical detection, could account for persistent concentrations of BCL2 mRNA as a target for the RaPP. In addition, the imaging modality used may not have been sensitive enough to detect a difference in ROI intensity in dogs of the present study at these time points. Limitations exist with conventional planar scintigraphy in that the unit generates less detail than do units that produce a 3-D image. Planar gamma cameras are the most abundant scintigraphy units in veterinary hospitals. Thus, single-headed gamma camera planar imaging was selected for the present study.

Results of the prospective imaging study and molecular analysis in the present study did not reveal a significant correlation between BCL2 mRNA concentration (determined in an affected lymph node excised for confirmation of B-cell lymphoma) and relative intensity (indicative of RaPP uptake) of any ROI at any time point examined. The CIs for the regression coefficient were very wide, indicating a lack of statistical power in this aspect of the study. Further studies are needed before it can be determined whether BCL2 mRNA concentrations are correlated with increased RaPP uptake or clinical outcome. These studies will likely require a more sensitive imaging technique, a larger number of dogs, and improved clearance of the imaging agent from the urinary system during image acquisition.

The lack of correlation between ROI intensity indicative of RaPP uptake and BCL2 mRNA expression in the present study may be related to several other factors. First, the amount of BCL2 mRNA template may be small in lymphoma cells in dogs. Second, variation in the concentration of the BCL2 mRNA was minimal among dogs of the present study. Third, the RaPP conjugate may have targeted somatostatin receptors without adequate targeting to BCL2 mRNA. Previous studies in mice21 have demonstrated that 111In-labeled RaPP is eliminated from tumor xenografts rapidly if no target is present. In a preliminary imaging experiment performed by the authors over a course of 1 to 48 hours, maximum urinary excretion occurred ≤ 1 hour (after IV injection in dogs), a sufficient amount of time for any nonspecifically targeted RaPP to be eliminated.

Minimal variation in BCL2 mRNA expression, such as that detected in dogs of the present study, may not be typical in human patients with B-cell lymphoma. In 1 study30 in humans, most (71%) diffuse large B-cell lymphoma tissues had BCL2 mRNA expression > 4-fold as high as that detected in germinal center B cells, when evaluated via complementary microarrays. A minority of diffuse large B-cell lymphomas (29%) had concentrations of BCL2 mRNA similar to that detected in germinal center B cells. This indicated that BCL2 may be important in some subtypes of this disease in humans.31 To our knowledge, a comparison of BCL2 mRNA expression between human and canine patients with lymphoma, by use of the same methods, has not been made.

The RaPP used in the study reported here has been confirmed to target human BCL2 mRNA in a laboratory setting.21 In that study, the RaPP was used to target BCL2-overexpressing Mec-1 cells implanted subcutaneously in immunodeficient mice. The cell line was derived from the peripheral blood of a human patient with B-cell CLL.31 In the present study, expression of BCL2 mRNA in lymphoid tissue from dogs with spontaneously occurring B-cell lymphoma was relatively low, compared with that reported in Mec-1 cells. To our knowledge, there are no reports describing BCL2 mRNA expression in healthy dogs.

It has been speculated that dogs with B-cell lymphoma have an abundance of the somatostatin receptors required for internalization of the type of RaPP used in the present study; this information was extrapolated from the knowledge of overexpression of somatostatin receptors in human lymphoma cells.32 Prior to initiation of the present study, the tissue-specific uptake of 111In-DOTA-Tyr3-octreotate, a peptide directed to somatostatin receptors, was evaluated in 3 dogs with B-cell lymphoma in a pilot studyp conducted at the University of Missouri. The compound used in that studyp differed from the conjugate used in the present study in that it lacked a PNA to target BCL2 mRNA. Data from the pilot study provided proof of principle that canine lymphoma cells express somatostatin receptors for molecular targeting.

The small sample size in the present study was considered a likely cause of the lack of significance between ROI intensity after RaPP administration and BCL2 mRNA expression. Although this aspect of the study was underpowered, inability to detect a significant correlation between ROI intensity and BCL2 mRNA expression may also have been caused by a lack of RaPP specificity to canine BCL2 mRNA. To further evaluate the specificity of the RaPP for targeting BCL2 mRNA, future studies would include scintigraphic imaging after administration of 111In-DOTA-Tyr3-octreotate conjugated to a scrambled PNA similar to that used in the previous study21 in mice.

A noninvasive imaging modality that provides information regarding BCL2 mRNA expression could potentially be used to provide prognostic information in evaluation of dogs with B-cell lymphoma. Data obtained could also provide a basis for suggesting specific adjuvant treatments, such as a combination of treatment with targeted antisense oligonucleotides and conventional chemotherapy, to improve response to treatment and survival times in these patients. Further research is needed to determine the clinical value of the RaPP used in the present study for detection and prognostication of B-cell lymphoma in dogs.

ABBREVIATIONS

BCL2

B-cell leukemia-lymphoma 2

CI

Confidence interval

CLL

Chronic lymphocytic leukemia

DOTA

1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid

PNA

Peptide nucleic acid

RaPP

Radiolabeled peptide nucleic acid–peptide conjugate

ROI

Region of interest

RT

Reverse transcriptase

TTP

Time to tumor progression

Tyr

Tyrosine

a.

Equistand II planar gamma camera, Diagnostic Imaging, Middlesex, NJ.

b.

Mirage version 54x, Diagnostic Services, Sussex, NJ.

c.

Open Biosystems, Huntsville, Ala.

d.

Wizard plus SV Minipreps DNA Purification System, Promega Corp, Madison, Wis.

e.

STAT-60 reagent, Tel-Test Inc, Friendswood, Tex.

f.

SUPERase-In, Applied Biosystems, Carlsbad, Calif.

g.

ND-1000 spectrophotometer, NanoDrop Technologies, Wilmington, Del.

h.

RNeasy MinElute cleanup kit, Qiagen, Valencia, Calif.

i.

Superscript III Reverse Transcriptase, Invitrogen, Carlsbad, Calif.

j.

TaqMan Gene Expression Assay, assay No. Cf02622425_m1, Applied Biosystems, Carlsbad, Calif.

k.

7500 Thermocycler, Applied Biosystems, Carlsbad, Calif.

l.

FAM fluorescent dye, Invitrogen, Carlsbad, Calif.

m.

ROX fluorescent dye, Invitrogen, Carlsbad, Calif.

n.

Sequence Detection Systems, version 1.2.3, Applied Biosystems, Carlsbad, Calif.

o.

R, version 2.12.2, R Foundation for Statistical Computing, Vienna, Austria. Available at: www.r-project.org/. Accessed Jan 6, 2011.

p.

Lewis M, Bryan J, Jia F, et al. Somatostatin receptor imaging of canine B-cell lymphoma (abstr), in Proceedings. 27th Annu Vet Cancer Soc Conf 2007;77.

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Contributor Notes

Supported by National Institutes of Health Grant No. CA103130 and Veterans Administration Grant No. BX000575.

Address correspondence to Dr. Lewis (LewisMic@missouri.edu).