Introduction
Lymphoma is the most common hematopoietic cancer in dogs. Multiple prognostic factors have been identified that help clinicians inform clients about the course of disease and projected outcome for their pets.1 Flow cytometric, immunohistochemical, and other molecular techniques have allowed veterinary clinicians to further subcategorize lymphoma into distinct phenotypes, each with its own biological behavior and prognosis.2,3,4 However, there remains a need for identification of additional prognostic factors to further characterize the disease, inform treatment choices, and predict outcome.
In human oncology, a recent focus has been the influence of the tumor microenvironment in supporting tumor progression. Tumor-enabling regulatory T cells and tumor-associated macrophages have been identified in multiple tumor types and extensively researched.5,6,7,8,9 More recently, the presence of tumor-associated neutrophils and peripheral neutrophilia has been associated with a poor outcome in human cancer patients. Neutrophils have historically been thought to play a protective role in cancer patients.10 However, tumor-associated neutrophils have been shown to promote tumor cell proliferation, encourage tumor angiogenesis, and produce reactive oxygen species that contribute to genomic instability in humans.11 They have been studied in multiple disease settings and are associated with a poor prognosis.12,13,14 Tumor-associated neutrophils in veterinary patients have not yet been studied, but they offer a theoretical explanation for poor patient outcomes.
Just as tumor-associated neutrophils have been shown to favor tumorigenesis in the tumor microenvironment, peripheral neutrophilia has been identified in several studies15,16,17,18,19,20,21,22,23,24 as a negative predictor of outcome in multiple human cancers. Importantly, peripheral neutrophilia may represent a poor prognostic indicator if those neutrophils indicate an inappropriate inflammatory response evoked by the cancer or represent circulating tumor-associated neutrophils. Human patients who have peripheral neutrophilia when lymphoma is diagnosed typically have a particularly aggressive form of disease, with shorter reported progression-free intervals and overall survival times.25,26,27 In veterinary medicine, Curran and Thamm28 reported neutrophilia as a poor prognostic indicator in dogs with lymphoma, but this finding was not further explored in that study. The precise reason for these associations has not yet been identified, but these findings necessitate further investigation.
The N:L ratio has also been used as a prognostic tool for human patients with lymphoma. This ratio serves as a representation of the inflammatory response evoked by the cancer, relative to the immune response, with higher ratios having been linked to poorer prognoses in human lymphoma patients.19,20,21 The N:L ratio was evaluated in a study29 of dogs with lymphoma, but no association between the N:L ratio and prognosis was found.
The objective of the study reported here was to determine whether, in dogs with naïve multicentric lymphoma treated with a CHOP-based protocol, the presence of peripheral neutrophilia was associated with PFST or the overall response rate (ie, percentage of dogs with a complete or partial remission). In addition, we wanted to determine whether the N:L ratio at the time of initial diagnosis would be significantly associated with PFST. Our hypotheses were that dogs with peripheral neutrophilia at the time of initial diagnosis of lymphoma would have a shorter PFST and decreased overall response rate and that the N:L ratio would be inversely associated with PFST.
Materials and Methods
Study population
The medical records database at the University of Wisconsin-Madison Veterinary Medical Teaching Hospital was searched to identify dogs examined between 2008 and 2018 that had multicentric lymphoma with neutrophilia at the time of initial diagnosis. For the purposes of this study, neutrophilia was defined as a neutrophil count higher than the upper reference limit for the laboratory where the CBC was performed. Dogs were included in the study only if they had not previously received any chemotherapeutic drugs or corticosteroids. Dogs with other severe, life-threatening disease processes or primarily extra-nodal lymphoma were excluded.
For dogs included in the study population, information obtained from the medical records consisted of signalment, immunophenotype (if available), results of staging tests (thoracic radiography, abdominal ultrasonography, bone marrow aspiration, urinalysis, echocardiography, and lymph node biopsy or extirpation), clinical stage (I through V), substage (a or b), clinical signs at the time of initial diagnosis, neutrophil count, lymphocyte count, treatment protocol, best response to treatment, date of disease progression or death, comorbidities, and medications being administered at the time of diagnosis.
Historical control population
A historical control population of 37 dogs with a neutrophil count within reference limits at the time of initial diagnosis of multicentric lymphoma was included to collectively represent the PFST of patients without neutrophilia treated with a CHOP-based protocol. All patients in this group had been treated with a 12-week CHOP-based protocol (CHOP 12) or a 19-week CHOP-based protocol (CHOP 19) as part of another study30 between December 2015 and December 2017. Although patients were enrolled prospectively for that study, patient information was collected retrospectively for use in the present study. As was the case for the study population, dogs were included in the historical control population only if they had not received any chemotherapeutic drugs or corticosteroids prior to the first dose of chemo-therapy for the CHOP protocol, and dogs with other severe, life-threatening disease processes or primarily extranodal lymphoma were excluded.
For dogs included in the historical control population, information similar to that for the study population was collected, except that information on clinical signs at the time of initial diagnosis, comorbidities, and medications being administered at the time of diagnosis was not collected as part of the previous or present study.
Data analysis
For both populations, immunophenotype, when available, was determined by immunocytochemical or immunohistochemical methods, a PCR assay for antigen receptor rearrangement, or flow cytometry. Stage was determined in accordance with World Health Organization guidelines. Substage was determined on the basis of clinical signs present at the time of initial diagnosis, as assessed by the attending clinician. Best response to treatment was defined as complete remission, partial remission, progressive disease, or stable disease. Dogs were considered to have had complete remission if all peripheral lymph nodes were palpably normal; partial remission if there was a ≥ 30% decrease in the sum of the longest diameter for all peripheral lymph nodes, compared with baseline measurements; progressive disease if there was a ≥ 20% increase in the sum of the longest diameter for all peripheral lymph nodes, compared with baseline measurements; and stable disease if they did not meet the criteria for complete remission, partial remission, or progressive disease. Overall response rate was defined as the percentage of dogs that had a complete or partial remission. The PFST was defined as the time from initiation of treatment to development of progressive disease, as determined by physical or cytologic examination of the lymph nodes, or death due to any cause.
Statistical analysis
The PFST and N:L ratio were the primary end points of the study. Overall survival time was not evaluated because overall survival time for dogs is heavily biased by owner beliefs and preferences regarding rescue therapy and euthanasia. Patients in the study population were grouped on the basis of treatment protocol as selected by the owners under the guidance of the overseeing oncologist. Median PFST in each group was calculated by means of the Kaplan-Meier method. Median PFST was also calculated for the historical control population.
For all dogs, log-rank analysis was used to compare PFST between groups when dogs were grouped on the basis of neutrophilia status (present vs absent), immunophenotype (B cell vs T cell), stage (1 through 4 vs 5), substage (a vs b), and treatment duration (CHOP 12 vs CHOP 19). For patients with neutrophilia, log-rank analysis was used to compare PFST between groups when dogs were grouped on the basis of treatment protocol (prednisone alone vs prednisone, l-asparaginase vs rabacfosadine vs doxorubicin, and lomustine vs CHOP 12 vs CHOP 19 vs other), stage (1 through 4 vs 5), substage (a vs b), and immunophenotype (B cell vs T cell). Other treatments included indenoisoquinoline (administered as part of a clinical trial) and a combination of doxorubicin and toceranib. A t test was used to evaluate for differences in patient characteristics and treatment response rate between the 2 populations. Multivariate analysis was not performed owing to the small size of the study population.
The N:L ratio was calculated for all patients, and a Cox proportional hazard regression model was used to test for a significant relationship between the N:L ratio and PFST while taking covariates into consideration.
All statistical analyses were performed with a commercially available statistics program.a Values of P ≤ 0.05 were considered significant.
Results
Study population
Forty-seven dogs examined between 2008 and 2018 because of multicentric lymphoma had neutrophilia at the time of initial diagnosis and were considered for inclusion in the study. However, 17 dogs were excluded because the absolute neutrophil count at the time of diagnosis was not included in the medical record or because the diagnosis of multicentric lymphoma was not confirmed. The remaining 30 dogs included in the study all had naïve multicentric lymphoma, confirmed on the basis of cytologic or histologic findings, and neutrophilia at the time of initial examination.
Breeds included Golden Retriever (n = 9), Labrador Retriever (8), Standard Poodle (2), Beagle (2), Spaniel mix (2), and Boxer (2). There was 1 each of Bull Mastiff, Saint Bernard, Cavalier King Charles Spaniel, Rottweiler, and American Bulldog. Seventeen were castrated males, 8 were spayed females, 3 were sexually intact males, and 2 were sexually intact females. Median age was 8.25 years (range, 4 to 12 years). Median weight was 35.2 kg (range, 5.6 to 64.2 kg). The most common comorbidity was idiopathic epilepsy (n = 3). There was 1 patient each with subaortic stenosis, focal seizures, and cranial nerve VII neuropathy. Four patients had concurrent or previously diagnosed neoplasia. One patient was incidentally found to have a hepatic tumor (suspected to be hepatocellular carcinoma on the basis of cytologic findings) and an adrenal mass during staging for lymphoma. One patient was incidentally found to have mammary masses on examination and a history of a cutaneous mast cell tumor that had been incompletely excised > 1 year prior to presentation. Another patient had a history of digital melanoma treated with complete digital amputation > 1 year prior to presentation. The final patient had an incidentally noted gingival mass on examination that was suspected to be benign but was not biopsied. In these 4 patients, neutrophilia was suspected to be secondary to multicentric lymphoma because the neutrophilia resolved when the lymphoma was in remission.
Thirteen of the 30 (43%) patients had B-cell lymphoma, and 4 (13%) had T-cell lymphoma. In these patients, immunophenotype was determined by flow cytometry (n = 11), immunocytochemistry (3), a PCR assay for antigen receptor rearrangement (2), and immunohistochemistry (1). One of the 13 patients with B-cell lymphoma had both aggressive B-cell and indolent T-zone lymphoma; for statistical purposes, this patient was included with the B-cell lymphoma group. Immunophenotype was not determined in the remaining 13 (43%) patients. Five (17%) patients had intermediate- to large-cell lymphoma, and 22 (73%) patients had large-cell lymphoma; cell size was unknown for 2 patients. The remaining patient had small- to intermediate-cell lymphoma on cytologic examination but was included in the study because flow cytometry results were consistent with an aggressive phenotype. One (3%) patient was classified as stage II, 10 (33%) were classified as stage III, 11 (37%) were classified as stage IV, and 8 (27%) were classified as stage V. Eleven (37%) patients were classified as substage a at the time of diagnosis (stage II, n = 1; stage III, 5; stage IV, 4; and stage V, 1), and 19 (63%) were substage b (stage III, 5; stage IV, 7; and stage V, 7).
Sixteen of the 30 (53%) dogs in the study population were treated with a CHOP-based protocol. Median age for these dogs was 8.75 years (range, 5 to 14 years). There were 10 castrated males, 3 spayed females, 2 sexually intact males, and 1 sexually intact female. Seven dogs had B-cell lymphoma, and 4 had T-cell lymphoma; immunophenotype was unknown for the remaining 5 dogs. Five dogs were substage a, and 11 were substage b.
A CBC and serum biochemical panel were performed on all 30 patients. Thoracic radiography was performed on 20 patients, and abdominal ultrasonography was performed on 9. Other staging tests included urinalysis (n = 9), bone marrow aspiration (3), echocardiography (2), and lymph node extirpation (1). The most common clinical signs at presentation were hyporexia or anorexia (n = 15), lethargy (12), weight loss (6), and polyuria and polydipsia (5). Other clinical signs at presentation included vomiting (n = 4), panting (4), diarrhea (4), facial swelling (3), increased respiratory rate and effort (3), alopecia (2), restlessness (2), pendulous abdomen (1), ventral edema (1), dysphagia (1), syncope (1), stertor (1), blindness (1), hypersalivation (1), ataxia (1), muscle wasting (1), nasal discharge (1), rectal prolapse (1), and hyphema (1).
Median PFST for the 30 dogs with neutrophilia, regardless of treatment protocol, was 55.5 days (range, 0 to 296 days; Table 1). Treatment protocol was significantly (P = 0.02) associated with PFST, with the greatest difference between dogs treated with prednisone alone and dogs treated with a CHOP-based protocol. However, many treatment groups consisted of small numbers of patients. Stage (P = 0.07), substage (P = 0.6), and immunophenotype (P = 0.06) were not significantly associated with PFST.
Median (range) PFST for dogs with naïve multicentric lymphoma that did or did not have neutrophilia at the time of initial diagnosis, grouped on the basis of treatment.
| Treatment | Dogs with neutrophilia | Dogs without neutrophilia | ||
|---|---|---|---|---|
| No. of dogs | PFST (d) | No. of dogs | PFST (d) | |
| All dogs | 30 | 55.5 (0–296) | 37 | 184.5 (23–503) |
| Prednisone | 3 | 0 (0–105) | 0 | NA |
| Prednisone and L-asparaginase | 3 | 13.5 (0–27) | 0 | NA |
| Rabacfosadine | 3 | 153 (0–264) | 0 | NA |
| Doxorubicin and lomustine | 2 | 143.5 (47–240) | 0 | NA |
| Other | 3 | 63 (23–70) | 0 | NA |
| All CHOP | 16 | 70 (0–296) | 37 | 184.5 (23–503) |
| CHOP 12 (stage I through IV) | 2 | 211.5 (0–296) | 17 | 140 (23–503) |
| CHOP 12 (stage V) | 0 | NA | 6 | 128 (23–404) |
| CHOP 19 (stage I through IV) | 11 | 133 (0–264) | 12 | 255.5 (77–405) |
| CHOP 19 (stage V) | 3 | 44 (30–50) | 2 | 259 (174–344) |
Other treatments included indenoisoquinoline (administered as part of a clinical trial) and a combination of doxorubicin and toceranib.
NA = Not applicable.
Historical control population
Forty-seven patients had been enrolled in the previous study30; however, 10 of these dogs had neutrophil counts outside reference limits and were excluded from the present study. Breeds included in the present study were Labrador Retriever (n = 6), Golden Retriever (4), Portuguese Water Dog (3), Corgi (3), Border Collie (2), and Boxer (2). There was 1 each of Jack Russell Terrier, Mastiff, Pug, Smooth Coated Fox Terrier, Husky mix, Rhodesian Ridgeback, Beagle, Springer Spaniel mix, Catahoula Leopard Dog, Old English Sheepdog, German Shepherd Dog, Rottweiler, Bulldog, Miniature Schnauzer, Doberman Pinscher, Flat-Coated Retriever, and Shih Tzu. There were 14 spayed female dogs, 19 castrated male dogs, 2 sexually intact female dogs, and 2 sexually intact male dogs. Median age at the time of diagnosis was 7.5 years (range, 3.5 to 13.25 years). Median weight at the time of diagnosis was 28.5 kg (range, 4.4 to 49.8 kg).
Twenty-five (68%) control patients had B-cell lymphoma, and 7 (19%) had T-cell lymphoma. Immunophenotype was unknown for 5 (14%) patients. All 37 patients had large-cell lymphoma. Twenty-two (59%) patients were classified as stage III, 7 (19%) were classified as stage IV, and 8 (22%) were classified as stage V. Twenty-seven (73%) patients were classified as sub-stage a (stage III, n = 20; stage IV, 3; and stage V, 4), and 10 (27%) were classified as substage b (stage III, 2; stage IV, 4; and stage V, 4). All patients had a CBC and serum biochemical panel performed. Thirty-one underwent thoracic radiography, and 5 underwent abdominal ultrasonography. Information on clinical signs and comorbidities was not available for the control population.
Comparisons of study and historical control populations
The percentage of substage b patients in the study population (19/30 [63%]) was significantly (P = 0.002) greater than the percentage of substage b patients in the historical control population (10/37 [27%]). Other patient characteristics did not differ significantly between the 2 populations.
Overall, 53 patients were treated with a CHOP-based protocol (16 in the study population and 37 in the historical control population). For these dogs, neutrophilia status (present vs absent) was significantly (P = 0.007) associated with PFST, with median PFST for dogs with neutrophilia at the time of initial diagnosis (median, 70 days; range, 0 to 296 days) substantially shorter than that for dogs without neutrophilia (median, 184.5 days; range, 23 to 503 days; Figure 1). Immunophenotype was also significantly (P = 0.02) associated with PFST; patients with T-cell lymphoma had a median PFST of 92 days (n = 11; range, 30 to 371 days), compared with 232 days (31; range, 0 to 503 days) for patients with B-cell lymphoma. Stage (P = 0.5), substage (P = 0.08; Figure 2), and treatment duration (CHOP 19 vs CHOP 12; P = 0.7) were not significantly associated with PFST.
Kaplan-Meier curves of PFST for dogs with naïve multicentric lymphoma that did (n = 16; blue) or did not (37; red) have neutrophilia at the time of initial diagnosis and were treated with a CHOP-based protocol. Shaded areas represent 95% CIs.
Citation: Journal of the American Veterinary Medical Association 259, 5; 10.2460/javma.259.5.494
Kaplan-Meier curves of PFST for dogs with naïve multicentric lymphoma classified as substage a (n = 32; red) or b (21; blue) at the time of initial diagnosis that were treated with a CHOP-based protocol. Shaded areas represent 95% CIs.
Citation: Journal of the American Veterinary Medical Association 259, 5; 10.2460/javma.259.5.494
Best response to treatment (complete remission, partial remission, stable disease, or progressive disease) was recorded for the study and historical control populations. For the study population, 15 (50%) patients had a complete remission, 4 (13%) had a partial remission, 4 (13%) had progressive disease, and 2 (7%) had stable disease; best response was unknown for 5 patients. Overall response rate for this group was 63% (19/30). For the 16 dogs with neutrophilia treated with a CHOP-based protocol, 10 had a complete remission, 2 had a partial remission, 1 had stable disease, and 1 had progressive disease; best response was unknown for 2 patients. Overall response rate for this group was 75% (12/16). For the historical control population, 33 of 37 (89%) patients had a complete remission, 3 (8%) had a partial remission, and none had progressive or stable disease; best response was unknown for 1 patient. Overall response rate for this group was 97% (36/37). Overall response rates were significantly (P = 0.01) different between groups.
N:L ratio
The median N:L ratio for all 67 patients was 5.67 (range, 0.27 to 92.33). The median PFST for patients with an N:L ratio at or above the median was 148 days (range, 0 to 503 days), compared with a median PFST of 142.5 days (range, 2 to 388 days) for patients with an N:L ratio below the median. The median N:L ratio for the 30 patients with neutrophilia was 9.7 (range, 1.44 to 92.33), whereas the median N:L ratio for the 37 patients without neutrophilia was 4.43 (range, 0.27 to 20.75). Cox proportional hazard regression analysis did not reveal a significant (P = 0.67) association between N:L ratio and PFST.
Discussion
Results of the present study confirmed our hypothesis that for dogs with naïve multicentric lymphoma treated with a CHOP-based protocol, the presence of peripheral neutrophilia at the time of initial diagnosis was associated with a shorter PFST and lower overall response rate (ie, percentage of dogs with a complete or partial remission), compared with values for dogs without peripheral neutrophilia. Specifically, for dogs treated with a CHOP-based protocol, median PFST for the 16 dogs with neutrophilia at the time of initial diagnosis (median, 70 days; range, 0 to 296 days) was significantly (P = 0.007) shorter than that for the 37 dogs without neutrophilia (median, 184.5 days; range, 23 to 503 days). Additionally, the overall response rate for dogs with neutrophilia (12/16 [75%]) was significantly (P = 0.01) lower than the rate for dogs without neutrophilia (36/37 [97%]). However, when all dogs in the study and control populations were considered together, the N:L ratio at the time of diagnosis was not significantly associated with PFST.
Neutrophils are typically thought of as the first responders of the innate immune system, playing important roles in the control of infection and tissue damage. However, the human literature has identified a subset of neutrophils, termed tumor-associated neutrophils, that have been shown to play protumoral rather than protective roles.10,11,31 These tumor-associated neutrophils have been shown to contribute to tumor angiogenesis, proliferation, and invasion by altering components of the extracellular matrix32,33 and releasing cytokines that increase expression of vascular endothelial growth factor.34 This is important because both tumor angiogenesis35 and modification of the extracellular matrix36 have been recognized to contribute to lymphomagenesis. Although their roles are not yet fully understood, there is a strong case for the influence of tumor-associated neutrophils in the tumor microenvironment in a wide variety of human cancers.13,14,34 Importantly, tumor-associated neutrophils have been shown in human medicine to play a role not only in the tumor microenvironment but also in the peripheral circulation,37 which may explain why peripheral neutrophilia may be a negative prognostic factor for PFST in patients with cancer. This link has yet to be made in the veterinary setting, and as such, investigation of the roles of tumor-associated neutrophils is warranted.
Another possible explanation for the poorer prognosis associated with neutrophilia could be the release of specific cytokines by lymphoma cells leading to a generalized inflammatory and bone marrow response that results in systemic effects and accelerated growth of neoplastic cells. This could be further investigated by prospectively assessing differences in cytokine expression between dogs with lymphoma that do or do not have neutrophilia. Neutrophilia may have also been associated with a shorter PFST in the present study as a result of increased disease burden and extended chronicity of disease. Indeed, the percentage of substage b patients in the study population (19/30 [63%]) was significantly (P = 0.002) greater than the percentage of substage b patients in the historical control population (10/37 [27%]), and the percentage of dogs in the study population with stage V disease (8/30 [27%]) was higher than the percentage in the historical control population (8/37 [22%]), although not significantly so. Owing to the small population sizes, we were unable to perform statistical analyses to determine whether neutrophilia status (present vs absent) was associated with stage or sub-stage. However, our results may suggest that the presence of neutrophilia may represent a more clinically aggressive disease phenotype.
Because neutrophils are key regulators of the inflammatory response, a case to link the presence of a high number of neutrophils in the peripheral circulation with an increased prevalence of clinical signs can be logically made. Under normal circumstances, neutrophils can contain the inflammatory response to a locality within the body. They have various granules, among other constituents, that contain antimicrobial proteins, proteases, and components of the respiratory burst that work in concert to orchestrate a contained immune response.38 An inappropriate production of neutrophils from the bone marrow, dysregulation of existing neutrophils, or the presence of tumor-associated neutrophils may lead to widespread neutrophil degranulation and release of potentially cytotoxic agents that result in substage b.38,39 In the present study, PFST was not significantly associated with substage. Nevertheless, prospective analysis with larger patient populations is needed to further evaluate this.
To our knowledge, the present study represented the first veterinary study to compare the N:L ratio between patients that had multicentric lymphoma with and without neutrophilia at the time of diagnosis. Although the N:L ratio differed between dogs with and without neutrophilia in the present study, the N:L ratio was not significantly associated with PFST. Furthermore, median PFSTs were similar between dogs with N:L ratios at or above the median and dogs with N:L ratios below the median. This was similar to the findings of Mutz et al,29 who also did not find an association between the N:L ratio and prognosis for dogs with lymphoma. In contrast, the N:L ratio has been found to be prognostic in numerous human cancer types. This ratio can be viewed as a representation of the systemic inflammatory response evoked by the tumor relative to the host immune response. Therefore, a higher ratio may be caused by a more malignant neoplastic condition that not only incites tissue damage and inflammation via cytokines but also causes immunosuppression of the host. The population of neutrophils in the peripheral circulation at the time cancer is diagnosed is still unclear, and cytokine-based studies are needed to determine whether neutrophils in the peripheral circulation have pro- or antitumoral effects. If the population of neutrophils represents tumor-associated neutrophils, then we should consider the idea that high peripheral neutrophil counts may be indicative of tumor progression. It is possible that a correlation between the N:L ratio and prognosis does exist in dogs with lymphoma, but a larger study population of homogeneously treated patients may be needed to demonstrate this. It is also possible that lymphomas that cause neutrophilia in dogs may also cause concurrent increases in the numbers of other WBCs, including lymphocytes. This could theoretically be caused by the release of generalized cytokines that act on the bone marrow in general, affecting both cell lineages, or by stimulation of both lineages by neoplastic co-opted leukocytes such as tumor-associated neutrophils and T-regulatory cells.
We recognize that peripheral neutrophilia may be caused by a variety of infections, chronic inflammatory disease processes, stress, and medications, especially glucocorticoids. However, patients in the study, especially patients with neutrophilia, had not yet been treated with glucocorticoids. Nevertheless, we must consider the possibility that other patient factors may have led to an increased peripheral neutrophil count, decreased peripheral lymphocyte count, or both, thereby skewing the N:L ratio. There were 4 patients in our study population that had previously or concurrently diagnosed or suspected neoplastic conditions, including mast cell tumors, melanoma, mammary tumors, hepatocellular carcinoma, a gingival mass, and an adrenal mass, and we recognize that this could have represented another cause of neutrophilia in these patients. However, for all of these patients, neutrophilia resolved with a successful response to chemotherapy. Furthermore, for a number of these patients, the other cancers had been previously treated with surgery, and there was no evidence of disease at the time of lymphoma diagnosis. Therefore, it seems most likely that the neutrophilia observed in these patients was related to their lymphoma diagnosis.
Several other findings of the present study were noteworthy. Patients in the historical control population with stage I through IV disease treated with CHOP 12 had a shorter median PFST (140 days; range, 23 to 503 days) than did those treated with CHOP 19 (255.5 days; range, 77 to 405 days), as was reported in a previous publication.30 For the study population of dogs with neutrophilia, treatment was found to be significantly associated with PFST, with the greatest difference in PFST between patients treated with prednisone alone versus those treated with a CHOP-based protocol; however, no difference was found in PFST between those treated with CHOP 12 versus CHOP 19 in this population. Although these findings make intuitive sense, they were severely limited by the small patient numbers, especially when patients were grouped by treatment. Stage of disease (stages I through IV vs stage V) was not significantly associated with PFST, consistent with a previous study.40 Immunophenotype (B cell vs T cell) was found to be significantly associated with PFST in the historical control population but not in the study population, although, again, this could have represented a type 2 error because of the small patient population size. Substage was also not found to be significantly associated with PFST, also likely because of limitations of the population size.
A large proportion of patients in our study population (13/30) did not have immunophenotyping performed, and it is possible that this lack of immunophenotyping skewed our results. Reported PFSTs for patients with nonindolent, nodal, T-cell lymphoma treated with CHOP-based protocols range from 5841 to 20042 days, with most studies reporting survival times toward the lower end of that range. Although we cannot completely rule out the possibility that PFST in our study population may have been biased as a result of having more patients with T-cell lymphoma, in general, a minority of patients with multicentric lymphoma (21% to 41%)1 have T-cell lymphoma. In addition, the group of patients with neutrophilia that did not undergo immunophenotyping included only 1 patient representing a breed predisposed to T-cell lymphoma (Boxer), and no patients had hyper-calcemia at presentation.
There were several limitations to our study inherent to its retrospective nature, such as variations in classification of substage, staging tests, and treatment. Subjectivity and variation in classification of substage have been demonstrated in canine lymphoma patients, even in a prospective study.43 Defined objective criteria to classify substage may be beneficial for future research. Because our institution represents a tertiary referral hospital, many patients had undergone initial clinicopathologic testing by another veterinarian at the time of presentation. Therefore, values from multiple diagnostic laboratories were used. To define neutrophilia, we chose to use the reference interval of whatever laboratory or hematologic machine was used for the initial analysis. As previously discussed, another limitation was the small patient population evaluated. Further analysis with a larger patient population, ideally in a prospective manner, is warranted.
In conclusion, our findings indicated that neutrophilia at initial presentation may suggest a poorer prognosis for canine patients with naïve multicentric lymphoma, compared with the prognosis for patients without neutrophilia. We believe that further exploration into the role of neutrophils in and outside of the tumor microenvironment of cancer-bearing patients is warranted, including attempts to specifically identify tumor-associated neutrophils and application of cytokine analysis to a prospective population of patients.
Acknowledgments
No third-party funding or support was received in connection with this study or the writing or publication of the manuscript. The authors declare that there were no conflicts of interest.
The authors thank Tedward Erker, statistical consultant for the University of Wisconsin-Madison College of Agricultural and Life Sciences, for assistance with statistical analyses.
Footnotes
R: A language and environment for statistical computing, R Project for Statistical Computing, Vienna, Austria. Available at: www.R-project.org/. Accessed Aug 21, 2019.
Abbreviations
| CHOP | Cyclophosphamide, doxorubicin, vincristine, and prednisone |
| N:L | Neutrophil-to-lymphocyte |
| PFST | Progression-free survival time |
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