Are B‐symptoms more reliable prognostic indicators than substage in canine nodal diffuse large B‐cell lymphoma

Abstract In humans B‐symptoms refer to systemic symptoms of lymphoma such as fever, weight loss, and night sweats and influence the prognosis of patients. In canine lymphoma, substage B is used to describe any clinical sign observed. Aim of the retrospective study was to compare the prognostic value of substage B with B‐symptoms to predict treatment response and survival in canine nodal diffuse large B‐cell lymphoma. Affected dogs treated with CHOP chemotherapy between 2008 and 2019 were included. B‐symptoms were defined by weight loss greater than 10% of normal weight, fever and the occurrence of unexplained resting tachypnoea substituted human night sweats. Substage B was defined as any symptoms but lymphadenopathy. Fifty‐five cases were included. B‐symptoms were present in 20/55 (36%) and substage B in 40/55 (74%) patients. No significant associations between B‐symptoms or substage B and weight, sex, breed, WHO stage and lymphoma grade were found. Treatment response was negatively associated with both substage B (P = .02) and B‐symptoms (P = .001). B‐symptoms significantly decreased progression free survival (PFS) (95 vs 330 days, P = .001) and lymphoma specific survival (LSS) (160 vs 462 days, P = .001). Data showed that B‐symptoms might be a more reliable prognostic indicator than substage B in canine nodal diffuse large B‐cell lymphoma. Prospective studies assessing B‐symptoms in a larger cohort of patients and in other common lymphoma types are warranted. The abstract was presented at the fourth meeting of the European Canine Lymphoma Network Group in Lugano, 22 June 2019 and published in the proceeding of the meeting on the page 26.


| INTRODUCTION
Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma in humans and dogs. Approximately 50% of all newly diagnosed cases and more than 80% of aggressive lymphomas are DLBCL. 1,2 Recent insights into the pathogenesis of DLBCL suggest that it is a heterogeneous group rather than a single entity. 1 Different morphologic variants are recognized, a variety of molecular and genetic abnormalities are present, and patients exhibit a wide range of clinical presentations and outcomes. 3 The CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisolone) chemotherapy protocol has been the mainstay of therapy for several decades. 4 Attempts to improve outcomes with more aggressive chemotherapy failed to show additional benefit. 5,6 The recent development of chemo-immunotherapy led to transformation of treatment practices in veterinary and human oncology and has improved outcome. 4,7 Although DLBCL is one of the most common lymphoma subtypes in dogs and it is in most cases highly responsive to chemotherapy, not every dog responds with complete remission to treatment. 4 Early identification of poor-risk patients may allow for alternate treatment strategies to be considered.
One of the most widely reported prognostic indicators is lymphoma substage, which has been correlated with complete treatment response (CTR), progression-free survival (PFS) and lymphomaspecific survival (LSS) in several studies. 8,9 The World Health Organization TNM Classification of Tumours of Domestic Animals details has stated simply substage A as the absence and substage B as the presence of clinical signs. 10 A recent study used a survey to query veterinary oncologists on the clinical criteria to define substage. 11 Gastrointestinal, constitutional, respiratory, neurologic, metabolic and nutritional variables were pivotal in assigning clinical substage. For most factors, a mild to moderate severity of clinical signs was sufficient for substage B designation. 11 However, any unrelated clinical sign could be interpreted as substage B rendering this system less reliable.
In human oncology, presence of B-symptoms is used to refer to systemic clinical signs of lymphoma as fever, weight loss, and night sweats and this classification influences prognosis. 12 Aim of our retrospective study was to compare the ability of substage B vs the presence of B-symptoms to predict treatment response and survival in canine nodal DLBCL (nDLBCL). We hypothesized that the presence of B-symptoms is superior to substage B in predicting outcome.

| Inclusion criteria
Previously treatment-naïve dogs diagnosed with nDLBCL at the Vetmeduni Vienna (Austria) and the Veterinary University Brno (Czech Republic) between 2008 and 2019 were included in this retrospective study. Fifty-five dogs from 113 patients with diagnosed lymphoma met the inclusion criteria: (a) confirmed nDLBCL by histopathology and immunohistochemistry, (b) no prior oncologic treatment including corticoids, (c) a standardized first-line chemotherapy CHOP (combination of cyclophosphamide, doxorubicin, vincristine, and prednisolone), and (d) a standardized clinical staging at the time of the initial diagnosis (at least based on peripheral blood analyses, thoracic radiographs, and abdominal ultrasound).

| Medical records review
Medical record data were reviewed and patient characteristics (age, breed, sex, weight), history, clinical symptoms, results of lymphoma staging, classification and grading, CTR and survival (PFS and LSS) were recorded. LSS was calculated from the date of diagnosis to the date of death related to the lymphoma. PFS was calculated from the date of diagnosis to the date of disease progression or recurrence.
Follow-up information was obtained from medical records and by phone conversations with referring veterinarians and pet owners.
Since 2016 a lymphoma-related questionnaire was used to assess history and signalment of patients. Dogs with primary extranodal or disseminated DLBCL were excluded from the study. Extranodal DLBCL was defined as lymphoma arising primarily outside of lymph nodes and spleen. Disseminated DLBCL was characterized as lymphoma involving broad spectrum of nodal and extranodal sites without evidence of primary origin. 13 Dogs that were alive at the end of study or lost to follow-up were censored at the date of their last contact.

| Lymphoma staging and classification
Medical records were explored for staging data including assessment of tumour extent (peripheral blood analyses, thoracic radiography, abdominal ultrasonography, fine-needle aspiration of liver, spleen and bone marrow and final stage based on the current World Health Organization (WHO) classification. 10 According to the published consensus of the Veterinary Comparative Oncology Group (VCOG) on response evaluation criteria for peripheral nodal lymphoma in dogs, a bone marrow aspirate was only requested if there was initially a clinical indication in the peripheral blood count/smear (cytopenia of unknown origin and/or lymphocytosis). 14 Ultrasonographic changes in homogeneity as well as echogenicity of liver and spleen were considered positive for lymphoma despite published literature. 15 Cytological samples of liver and spleen were considered as positive for lymphoma involvement if more than ≥5% of nucleated cells consisted of large lymphocytes. 15,16 Bone marrow cytology was recorded as positive if more than 3% of large lymphocytes or aggregates of lymphocytes were observed. 17 Dogs without peripheral blood analyses, thoracic radiographs and/or abdominal ultrasonography were excluded from the study.

| Definition of substage and B-symptoms
Substage A was defined as the absence of clinical signs but peripheral lymphadenopathy and substage B as presenting with any clinical signs of illness including gastrointestinal, constitutional, respiratory, neurologic, metabolic and nutritional symptoms. 11 B-symptoms were defined by unintentional weight loss of >10% of normal body weight over a period of six months or less, fever >39 C for more than three days without any evident cause other than lymphoma and human drenching night sweats were substituted with a presence of unexplained tachypnea at rest, that was defined as a higher frequency (panting) of breathing observed by owners in rest without any evident cause other than lymphoma. 12 B-symptoms have been in use for human lymphoma for decades and their accurate origin cannot be recalled from the published literature. The first documented adoption was established together with the Ann-Arbor system. 18 As there is no published clinical experience with B-symptoms in veterinary literature, we adopted criteria being long-term in use for humans. The defining features of DLBCL were the diffuse arrangement of sheets of neoplastic B cells and the uniformly large nuclei (>two red cells in diameter) and a scant cytoplasm of neoplastic cells. Nuclei were usually round or rarely cleaved or indented. 19 Lymphoma with 0 to 5 mitoses per high power field (HPF, 40x objective) were graded as grade I (low grade), with 6 to 10 mitoses per HPF as grade II (intermediate grade) and with more than 10 mitoses/HPF as grade III (high grade). 19

| Assessment of treatment response
Treatment response was evaluated by using the following VCOG criteria for response evaluation in peripheral nodal lymphoma in dogs: complete remission (CR), 100% reduction in size of all measurable disease; partial remission (PR), >30% but <100% reduction in size of all measurable disease; stable disease (SD) <30% reduction in size of all measurable disease with no change in size or <20% increase in size of all measurable disease; and progressive disease (PD), >20% increase in size of all measurable disease or the appearance of any new lesion. 14 Response was evaluated at each therapeutic session and was required to last for at least 28 days as previously reported. 14 For statistical analysis, CTR was defined as 100% reduction in size of all measurable and non-measurable disease present at the end of the first-line treatment. Statistical analysis included the t-test for normally distributed data and the Mann-Whitney U-test for non-normally distributed or categorical data. Pearson r correlation was used to compare the relationship between linear-related variables, and Spearman rank correlation between nonparametric variables.

| Statistical analyses
PFS was calculated as the interval between initiation of treatment and disease progression or relapse, whereas LSS was measured as the interval between the day of primary diagnosis and lymphoma-related death. Dogs lost to follow-up or dead for lymphoma-unrelated causes, as well as those still in complete remission at the end of the study, were censored from survival analysis. CTR was defined as the achievement of complete remission during and at the end of induction chemotherapy.
Survival curves were analysed with the Kaplan -Meier method.
Survival times are presented as median (range). Differences between Kaplan -Meier curves were assessed by the log-rank test. For all statistical analyses, a P-value <.05 was considered to be significant. Variables found to display statistical significance at the 5% level were included in a multiple analysis using multiple Cox proportional hazard analyses method.

| RESULTS
Fifty-two dogs were included into this study. The median body weight of the dogs was 28 kg (range: 3-55 kg) and the median age at the time of the initial examination of 8.9 years (range: 3-15 years) ( Table 1).
Eleven dogs were mixed breed dogs, the remaining 44 dogs represen- Thoracic radiography and abdominal ultrasound were performed in all 55 dogs including liver and/or spleen aspirates in 12 cases (22%).
Bone marrow aspiration was performed in nine cases (16%) and revealed positive results in all cases. Splenic infiltration was recorded in 27 dogs (49%). The liver was considered infiltrated in 11 dogs (20%), as documented by sonographic changes and/or confirmative cytology.
Peripheral blood was involved in 10 dogs. In addition to these, 14 (25%) dogs had also extranodal involvement other than peripheral blood, bone marrow or liver, lung infiltrates were most frequently recorded (n = 9) followed by intraocular (n = 3), dermal (n = 2), central  The presence of B-symptoms was significantly associated with shorter PFS and LSS (P < .001 for both) (Table 3, Figure 1A,B). Median PFS for dogs with and without B-symptoms was 91 (SD ± 10.2 days) vs 332 days (SD ± 58.7 days) respectively (Table 3). Median LSS for dogs with and without B-symptoms was 162 (SD ± 7.8 days) vs 462 days (SD ± 77.5 days), respectively.
The presence of substage B was not associated with shorter PFS (P = .18) and LSS (P = .23) ( Table 3, Figure 2A, F I G U R E 1 A, Kaplan-Meier progression-free survival of the 55 canine patients with nDLBCL stratified according to the presence or absence of B-symptoms: Presence of B-symptoms was an independent predictor of inferior time to progression/tumour recurrence (P = .001). B, Kaplan-Meier lymphoma specific survival of the 55 canine patients with nDLBCL stratified according to the presence or absence of B-symptoms: Presence of B-symptoms was an independent predictor of inferior survival (P = .001) was 160 (SD ± 27.7 dogs) vs 240 days (SD ± 91.5 days) ( Table 3).
The presence of B-symptoms (P < .001), but not substage B  The published studies describing canine nDLBCL suggested that substage B may be associated with inferior survival. 8,16 The presence of B-symptoms in dogs with lymphoma has been historically concluded to be low and authors are not aware of any veterinary study proving its prognostic relevance in canine nDLBCL or any other lymphoma subtype according to WHO classification. 20 Our data indicate that the presence of B-symptoms is more likely to be of prognostic significance than substage B. Data suggests that One important limitation of our study was the absence of a detailed staging including liver, spleen and bone marrow aspiration in all dogs. This is in a contrast with published literature showing that an altered echogenicity may be due to causes different from lymphoma, and that normal-appearing organs may be still infiltrated. 15,21 Another limitation is the lack of a standardized rescue treatment protocol as a result of variability in clinician's and owner's preferences, which could have introduced differences in the investigated parameters.
All data regarding B-symptoms and substage B were retrieved from medical records and/or recent client communication.
Tachypnoea was defined as increased frequency of breathing in rest without any cause other than lymphoma. Based on the retrospective design of the study, interpretation bias cannot be ruled out completely. However, all dogs presenting with tachypnea displayed also one or two other B-symptoms (weight loss and/or fever) making its influence on presented results unlikely. Nevertheless, the respective relevance of these three signs within B-symptoms alone and their combinations shall be further investigated in prospective studies with canine nDLBCL. We will suggest to set a timeframe for tachypnoea at rest for at least 3 days.
Strengths of the study are the long-term follow-up of a welldefined group of dogs with nDLBCL treated with a standardized firstline chemotherapy and low number of patients lost from the follow-up.
In conclusion, this study demonstrated that B-symptoms may be a better prognostic indicator than substage B in canine nDLBCL.
B-symptoms remained an independent predictor of survival in the multiple regression analysis. Human studies proved that B-symptoms are associated with increased concentrations of acute phase proteins and some proinflammatory cytokines. 12,22 Future prospective studies assessing B-symptoms in larger cohort of DLBCL patients and their association with inflammatory proteins and cytokines are warranted.