Pet dogs most often spontaneously develop B-cell lymphoma [1], for which survival without treatment is measured in weeks or months. Anthracycline-based multidrug chemotherapy represents the cornerstone for the treatment of canine B-cell lymphoma across all disease stages; however, the majority of dogs eventually relapses and develops chemo-resistance, and cure is rarely achieved [2].

Immunotherapy represents a powerful way to treat cancer, and the last decade has witnessed unprecedented advancements in the understanding of both the molecular drivers of lymphomagenesis and mechanisms by which lymphoma circumvents anti-tumor immunity [3, 4].

In oncology, therapeutic vaccines have the theoretical ability to generate cytotoxic T-lymphocytes that reject cancer cells, and memory cells that prevent relapse [5, 6].

Active immunotherapy consisting of hydroxylapatite ceramic powder and HSPs purified from the dogs’ tumors (APAVAC®) has shown promising results in a pivotal placebo-controlled, randomized clinical trial in dogs with DLBCL [7]. Within the first few years of its introduction to the European market, the investigational use of APAVAC® was extended to indolent B-cell lymphomas with suggestive signals of potential usefulness [8].

The aim of this retrospective study was to compare the efficacy and safety of chemotherapy alone versus chemo-immunotherapy in dogs with multicentric B-cell lymphoma of various histotypes in 5 years of clinical experience. The survival advantage of chemo-immunotherapy was further assessed after stratifying dogs according to potential prognostic variables, in order to identify the best candidates for vaccine administration.

Pooling the available clinical data should provide an accurate evaluation of the efficacy of chemo-immunotherapy in canine B-cell lymphoma compared with traditional chemotherapy, posing the basis for future clinical trial design and informing human trials.

Medical records were reviewed for every consecutive canine patient with previously untreated multicentric B-cell lymphoma that presented to the Centro Oncologico Veterinario (Sasso Marconi, Italy) and Department of Veterinary Medicine (University of Milan, Italy) from 2013 to 2018. This study was prospectively conceived and clinical data on the efficacy and toxicity of treatments were analyzed retrospectively, thus the inclusion criteria were established a priori and all dogs were treated contemporaneously.

To be eligible for inclusion, dogs were required to undergo a complete initial staging work-up and, whenever possible, surgical removal of a peripheral LN to obtain a histopathological diagnosis and material for the vaccine generation (Additional file 1). Based on histopathology and immunohistochemistry (CD20, CD3), tumors were classified according to the modified WHO criteria [9].

Also, dogs were only included, if information regarding treatment and outcome were complete.

As this evaluation did not influence any therapeutic decision, approval by an Ethics Committee was not required. Owners gave their written informed consent to the use of clinical data.

The total population consisted of 300 dogs: 246 (82%) underwent lymphadenectomy, whereas in the remaining 54 (18%) a cytological and FC diagnosis only was obtained.

Within the histology group, DLBCL was the most common histotype (n = 148; 60.2%), followed by nodal MZL late stage (n = 49; 19.9%), FL (n = 25; 10.2%), Burkitt lymphoma (n = 10; 4.1%), SLL (n = 8; 3.3%), and lymphoblastic lymphoma (n = 6; 2.4%).

Within the cytology group, there were 50 (92.6%%) centroblastic/immunoblastic lymphomas, 2 (3.7%) lymphoblastic lymphomas, and 2 (3.7%) medium macronucleolated cell lymphomas.

Within the whole population, 148 (49.3%) dogs received chemotherapy alone and 152 (50.7%) chemo-immunotherapy.

Median TTP was 147 days (95% CI, 111–183) for dogs receiving chemotherapy and 244 days (95% CI, 218–270) for those receiving chemo-immunotherapy (P <  0.001). Median LSS was 220 days (95% CI, 157–243) for dogs receiving chemotherapy and 401 days (95% CI, 339–463) for those receiving chemo-immunotherapy (P < 0.001).

The three main histotypes are detailed in the following paragraphs.

Immunotherapy is increasingly acknowledged as an effective treatment for several canine cancers including, malignant melanoma, B-cell lymphoma, osteosarcoma, and brain tumors [7, 8, 14–22]. Studies in the field have unfortunately shown that not all dogs will have a substantial benefit, and it is incumbent upon clinicians to decide whether a dog is or is not a candidate for immunotherapy.

Herein, we present the results of a study aimed at comparing the outcomes of dogs with B-cell lymphoma receiving chemotherapy or chemo-immunotherapy. The primary goal of this study was to identify the candidate dogs that would benefit the most from chemo-immunotherapy in the standard daily clinical practice outside a trial setting. So far only two small clinical trials on chemo-immunotherapy have been reported [7, 8]. The current study ranks among the largest per number of dogs treated with the same product for a specific disease and confirms the efficacy and the good safety profile of this treatment.

It was herein confirmed that DLBCL is the most common histotype in dogs, followed by MZL and FL. By analyzing the whole population it was also confirmed that stage III disease is quite infrequent, whereas stage V was most commonly diagnosed (70.7%). It is likely that an accurate staging leads to stage migration, having prognostic and therapeutic implications, as documented by the current data.

Overall, the present study demonstrates a significant clinical benefit of immunotherapy. In particular, subgroup analysis of LSS, comparing treatment arms using a multivariable method, indicated the following.

It was found that immunotherapy conferred a survival benefit in the majority of DLBCL cases. However, dogs that benefited the most were those with stage V disease, no systemic symptoms, a high serum LDH, and not previously treated with steroids. Interestingly, if these characteristics were concurrent, vaccinated dogs had the highest survival advantage (480 vs 85 days, respectively).

If two or three of the abovementioned characteristics were present, the survival benefit for vaccinated dogs was still significant, although to a lesser extent (435 vs 190 days, respectively). If only one or none of the abovementioned characteristics were present, there was no demonstrable benefit of chemo-immunotherapy over chemotherapy, although these cases represented a minority (n = 22).

It could be hypothesized that the stimulation of the immune system may contribute to counteract the negative impact of well-known prognostic factors that commonly lead to a poor outcome if chemotherapy only is administered. While chemotherapy effects only last as long as the drugs remain in the body, immunotherapy can provide long-term protection against cancer due to the immune memory, overcoming the “honeymoon effect” provided in the short term by cytotoxic drugs, more over if negative prognostic factors are present. Chemotherapy fails when drug-resistant clones emerge, preventing tumor cell eradication [23]. It may be possible that dogs with negative prognostic factors, such as advanced disease stage and high LDH levels, are more susceptible of developing chemo-resistance [2, 24, 25]. In these dogs, chemo-resistant clones may be eliminated by cell-mediated immunotherapy because they can evade neither immune surveillance nor immune response, thereby providing a survival benefit.

Finally, the lower immunity in symptomatic dogs is a plausible explanation to the suboptimal response to immunotherapy, and specific immune responses may be abrogated by pre-treatment steroids.

These findings were also confirmed for the MZL cases: vaccinated dogs survived significantly longer than unvaccinated dogs (680 vs 160 days, respectively; P < 0.001) if three or four of the previously identified characteristics were present. If 2 to none of those were present, there was no significant benefit in administering chemo-immunotherapy over chemotherapy.

In dogs with FL, a survival benefit of vaccine-administration was demonstrated as well, however the population was too small to evaluate the potential inference of other prognostic factors.

Finally, it must be reminded that, although chemo-immunotherapy seems to provide better results than chemotherapy for the majority canine B-cell lymphomas, the 3-year survival rate remains largely unsatisfactory, ranging from 0 to 12%; whereas the largest survival benefit can be experienced in the short/medium-term period. It is currently unknown whether re-vaccination or preparation of a new vaccine starting from nodal relapses would improve long-term survival.

Our study has a number of limitations, such as the small number of dogs included in some of the categories and its retrospective nature. However, we have included consecutive dogs diagnosed following a uniform approach and treated with standardized protocols.

Third, the evaluation of the immune response by the DTH skin test, albeit clinically relevant, is operator-dependent. Standardized and quantitative analyses are warranted to reduce this constraint.

Last, LSS may have been influenced by tumor’s unrelated factors, including owners’ motivation and financial concern. However, almost all dogs in both treatment arms received at least one rescue protocol, thereby reducing the risk of bias.

Our study shows a response and survival benefit of the addition of active immunotherapy to chemotherapy in dogs with B-cell lymphoma, possibly due to a different and perhaps synergistic mechanism of action. In the future, the development of new effective immunotherapeutic strategies should take into account differences in immune microenvironment between different lymphoma molecular subtypes to find the best treatment for each patient. A large-scale, double-blinded, randomized, multi-institutional trial is essential for ascertaining the efficacy of the presently described active immunotherapy procedure and its clinical application.